Camptothecin derivatives

ABSTRACT

New camptothecin derivatives possessing either or both of high anti-tumor activity and slight toxicity, represented by the general formula: ##STR1## wherein X is H, CH 2  OH, COOH, an alkyl group, an aralkyl group or the grouping CH 2  OR 1  or COOR 2  wherein R 1  is an alkyl group or an acyl group and R 2  is a lower alkyl group, Y is H, OH or the grouping OR 3  wherein R 3  is a lower alkyl group or an acyl group, and Z is H or an acyl group, with the proviso that when X is CH 2  OH, an alkyl group or an aralkyl group, both Y and Z are H, that when X is the grouping CH 2  OR 1  or COOR 2 , Y is H, that when Y is OH, both X and Z are H, .[.and.]. that when Y is the grouping OR 3 , X is H, .Iadd.and that X, Y and Z are not each simultaneously hydrogen, .Iaddend.and water-soluble alkali metal salts thereof. These camptothecin derivatives are prepared by treating camptothecin with sulfuric acid and a persulfate or with sulfuric acid and a peroxide, if necessary, with an organic compound corresponding to the organic moiety of the substituent to be introduced directly into camptothecin, in an aqueous medium in the presence or absence of a transition metal ion, and optionally treating the resultant products, if necessary, after oxidation of the introduced substituent, with an alkylating agent or an acylating agent.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to new derivatives of camptothecin, an alkaloidpossessing anti-tumor activity (including carcinostatic activity), andto processes for preparation of such derivatives. More particularly,this invention relates to new camptothecin derivatives bearing hydroxyor a functionally converted hydroxy substituent in the 5-position or anorganic carbon substituent in the 7-position thereof and possessing atleast one of strong anti-tumor activity and low toxicity as well asprocesses for the preparation of such derivatives.

2. Description of the Prior Arts

Camptothecin is a cytotoxic alkaloid, isolated first by Wall and hisco-workers [J.Am. Chem. Soc. 88(1966), 3888] from leaves and barks ofCamptotheca accuminata (NYSSACEAE), a plant native to China, which has apentacyclic structure consisting of a fused ring system of quinoline(rings A and B), pyrroline (ring C), α-pyridone (ring D) and asix-membered lactone (ring E) and displays dextro-rotation due to theS-configuration of a tertiary hydroxy group in the 20-position. Earlierreports on the carcinostatic activity of camptothecin based oninhibitory activity toward an experimentally transplanted carcinoma suchas leukemia L-1210 in mice or Walker 256 tumor in rats [Chem. Rev.23(1973), 385; Cancer Treat. Rep., 60(1967), 1007] stimulatedsynthetical researches on camptothecin, but the subsequent biologicalevaluation in the reports indicated that this compound is highly toxicand consequently unusable as a chemotherapeutic agent. Because of hightoxicity, camptothecin itself is not utilized at present for clinicaltreatments except in China, but this compound is still one of the mostpotent substances with antitumor activity and is thus regarded asimportant in the aspect of a biological reagent capable of inhibitingselectively the biosynthesis of ribosomal and messenger RNA's withoutdisturbing the biosynthesis of mitochondrial, 4S or 5S RNA's [Nature(London), New Biol., 237(1972), 144].

Such earlier reports on the significant antitumor activity ofcamptothecin stimulated intensive interest in the total syntheses andchemical modifications of camptothecin. Many papers describe thesyntheses of dl-camptothecin, its intermediate derivatives, and(+)-20(S)-camptothecin; synthesis of (+)-20(S)-camptothecin(Dextro-rotary) is reported by E. J. Corey et al., in J. Am. Chem. Soc.40 2140 (1975). In addition, synthesis of dl-camptothecin is reported,for example, by J. C. Bradley et al., in J. Org. Chem. 41,699(1976) andby H. G. M. Walraven et al., in Tetrahedron 36, 321 (1980), the latterbeing a report on the latest synthesis of camptothecin. The naturalcamptothecin isolated from Camptotheca accuminata is known to be in thed-form. However, none of these reports refer to chemical modification ofthe original structure of camptothecin from the standpoint ofchemotherapeutic usage. The chemical modifications so far reported aremainly concerned with the rings D and/or E of camptothecin, but theresults of such modifications have revealed only failure in maintainingexpected carcinostatic activity and poor improvement in toxicity [J.Med. Chem., 19(1976), 675] .

From the chemotherapeutic point of view, it is of importance that thechemical modifications of camptothecin should be restricted in the ringsA, B and C without effecting any serious change in the whole skeletalstructure, especially in the rings D and E of the natural camptothecin,the latter rings D and E being conceivable to be one of the essentialstructural elements for the expression of the above mentioned biologicalactivity. Functionalization of a moiety containing the rings A, B and Cis little known, except for nitration of camptothecin in concentratedsulfuric acid under severe conditions conducted in China to obtain12-nitrocamptothecin after troublesome separation treatments from otherproducts. This 12-nitro derivative is then reduced to the corresponding12-amino derivative which is further subjected to diazotization andsubsequent hydrolysis or a Sandmeyer reaction to introduce a hydroxygroup, chlorine atom, cyano group or carboxyl group into the 12-positionof camptothecin [P. Pei-chuang et al.; Hau Hsueh Hsueh Pao, 33 (1975),71; Chem. Abstr., 84 (1976), 115629p]. According to this method,however, it takes four steps to prepare the 12-cyano derivative and fivesteps to prepare the 12-carboxy derivative from the starting naturalcamptothecin. Except for this method wherein a number of troublesomesteps are required for introducing a functional substituent into the12-position of camptothecin, there has not yet been known heretofore anychemical modification for introducing a functional substituent in thering A, B and/or C. The reason why introduction of a substituent intothe ring A, B and/or C of camptothecin is extremely difficult isprobably ascribable to poor solubility of camptothecin in ordinaryorganic solvents and to the nature of a nitrogen-containing heterocyclicring which refuses an ionic reaction, especially the so-calledelectrophilic reaction conventionally carried out on aromatic rings,such as the Friedel-Crafts reaction, Vilsmeier-Haack reaction or otheralkylation or acylation reactions.

Thus, there is still a great demand in this art for developing newderivatives of camptothecin possessing at least one of high anti-tumoractivity and very weak toxicity by chemically modifying naturalcamptothecin on its ring A, B and/or C in one step without effecting anychange in the structure of the rings D and E which are regarded to beindispensable for exhibiting the physiological activity.

BRIEF SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide newcamptothecin derivatives which are effective antitumor agents especiallyuseful for both injection and oral administration.

It is another object of the present invention to provide newcamptothecin derivatives which are strong in anti-tumor activity andpossess good absorbability in the living body with very low toxicity.

It is still another object of the present invention to provide processesfor the preparation of the new camptothecin derivatives.

It is a further object of the present invention to provide new means forintroducing subtituents into the ring B or C of camptothecin without anymodifications of the structure of the rings D and E of camptothecin.

It is still a further object of the present invention to provide the useof the new camptothecin derivatives as anti-tumor agents.

Other objects, features and advantages of the present invention willbecome apparent more fully from the following description.

DETAILED DESCRIPTION OF THE INVENTION

With an attempt to synthesize new camptothecin derivatives whilemaintaining the inherent anti-tumor activity with extremely reducedtoxicity, the present inventors have conducted research for replacingany of the hydrogen atoms existing in the rings A, B and C with asubstituent other than the hydrogen atom, paying careful attention tothe replacement lest any change should occur in the structure of therings D and E which are regarded to the indispensable for exhibiting thephysiological activity of camptothecin. As a result of the presentinventors' extensive research, it has been found surprisingly that ahydroxy group can be introduced into the 5-position and various organicgroups can be introduced into the 7-position of camptothecin whilekeeping the rings D and E unchanged, when a radical substitutionreaction in place of the conventionally employed ionic reactions isapplied to camptothecin in a dilute aqueous acidic solution. The presentinvention which established a general method for introducing afunctional substituent into a specific position of camptothecin is basedon the above finding. Thus, it now becomes possible for the first timeto prepare a series of new camptothecin derivatives in one step byintroducing a functional substituent into the 7-position in the ring Band into the 5-position in the ring C while keeping the fundamentalskeletal structure of the rings A, B, C, D and E and the functionalgroups therein unchanged during the substitution reaction.

In accordance with one embodiment of the present invention, there areprovided new camptothecin derivatives of the general formula: ##STR2##wherein X is H, CH₂ OH, COOH, an alkyl group, an aralkyl group or thegrouping CH₂ OR¹ or COOR² where R¹ is an alkyl group or an acyl groupand R² is a lower alkyl group, Y is H, OH or the grouping OR³ where R³is a lower alkyl group or an acyl group, and Z is H or an acyl group,with the proviso that when X is CH₂ OH, an alkyl group or an aralkylgroup, both Y and Z are H, that when X is the grouping CH₂ OR¹ or COOR²,Y is H, that when Y is OH, both X and Z are H, and that when Y standsfor the grouping OR³, X stands for H, as well as water-soluble alkalimetal salts thereof.

When X and R¹ each represent an alkyl group, they may be the same ordifferent and generally have 1-30 carbon atoms. In view of theavailability of alkylating reactants, the alkyl group has preferably1-18 carbon atoms. Preferable examples of the alkyl group includestraight or branched chain alkyl groups with 1-18 carbon atoms, such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl,n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, 2-ethylhexyl, n-nonyl,n-decyl, undecyl, dodecyl, myristyl, heptadecyl and octadecyl groups.When the alkyl groups are branched, the branched chains may be combinedtogether to form a cycloalkyl group, Illustrative of such cycloalkylgroups are, for example, cyclopentyl, cyclohexyl, cycloheptyl andcyclooctyl. When R² and R³ each represent a lower alkyl group, they mayalso be the same or different and usually have 1-8 carbon atoms. Asdescribed above, both lower alkyl groups R² and R³ may have straight orbranched chains and in the latter case the branched chains may becombined together to form a cycloalkyl group. Preferable examples of thelower alkyl group include a straight or branched chain alkyl group with1-4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, tert-butyl and cyclopropyl groups. Preferable examples of thearalkyl group include benzyl, phenethyl, phenylpropyl and1-naphthylmethyl.

In general, when the substituent X exists in the 7-position of the ringB, no substituent exists in the 5-position of the ring C, or in otherwords, Y is H. On the other hand, if the substituent Y exists in the5-position of the ring C, no substituent exists in the 7-position of thering B. When R¹ and R³ and Z are each an acyl group, they are usuallythe same but may be different. The acyl group is derived from analiphatic or aromatic carboxylic acid, a halogen-substituted homologthereof and an aliphatic or aromatic sulfonic acid. Illustrative of thealiphatic and aromatic carboxylic acids and sulfonic acids are, forexample, formic acid, acetic acid, propionic acid, butyric acid,isobutyric acid, valeric acid, caproic acid, caprylic acid, nonylicacid, decanoic acid, phenylacetic acid, phenylpropionic acid, succinicacid, trifluoroacetic acid, benzoic acid, methanesulfonic cid,ethanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid.

The camptothecin derivatives of this invention possess excellentpharmacological properties improved in at least one of the anti-tumoractivity and the toxicity properties. Illustrative of the typicalcamptothecin derivatives of the present invention are7-hydroxymethylcamptothecin, 5-hydroxycamptothecin, 20-O-acetyl-7-acetoxymethylcamptothecin, 7-acetoxymethylcamptothecin,7-succinoyloxymethylcamptothecin,20-O-trifluoroacetyl-7-trifluoroacetoxymethylcamptothecin,7-benzoyloxymethylcamptothecin, 7-propionyloxymethylcamptothecin,7-butyryloxymethylcamptothecin, 7-caprylyloxymethylcamptothecin,7-capryloxymethylcamptothecin, 7-isovaleryloxymethylcamptothecin,7-phenylacetoxymethylcamptothecin, camptothecin-7-carboxylic acid, ethylcamptothecin-7-carboxylate, 5-methoxycamptothecin, 5-butoxycamptothecin,5-acetoxycamptothecin, 20-O-acetyl-5-acetoxycamptothecin,5-benzoyloxycamptothecin, 7-methylcamptothecin, 7-ethylcamptothecin,7-propylcamptothecin, 7-butylcamptothecin, 7-heptylcamptothecin,7-nonylcamptothecin, 7-isobutylcamptothecin, 7-benzylcamptothecin,7-β-phenethylcamptothecin, 7-isopropylcamptothecin and7-cyclohexylcamptothecin.

The new camptothecin derivatives of the present invention are not onlylimited to those from the naturally occurring (+)-camptothecin butinvolve also those from the corresponding (-)- and dl-camptothecinssynthetically obtained.

As camptothecin itself carries a lactone ring as ring E, this lactonering is opened by the action of an alkaline reagent. Similarly, when thecamptothecin derivatives of the present invention are treated, forexample, with an alkali metal hydroxide or carbonate in a conventionalmanner at room temperature or at an elevated temperature, thederivatives can be converted into the corresponding alkali metal saltsuch as the sodium, potassium or lithium salt. These salts are allwater-soluble and are of course involved in the scope of this invention.These salts are easily converted again into the free form by the actionof an acid or in vivo. Thus, the pharmacological effect of thecamptothecin derivatives is not influenced by such treatments. Apreferable salt of the camptothecin derivatives is the sodium orpotassium salt.

In accordance with the present invention, there is also provided aprocess for the preparation of the camptothecin derivatives. In oneembodiment of the process, camptothecin derivatives of the generalformula: ##STR3## wherein X' is the grouping COOR⁴ or CH₂ OR where R⁴ isH or a lower alkyl group and R is H, an alkyl group or an acyl group,and Z is H or an acyl group, as well as water-soluble alkali metal saltsthereof, are prepared by subjecting camptothecin to a radical reactionwith a hydroxymethyl compound of the general formula:

    A--CH.sub.2 OH                                             [II]

wherein A is H, COOH or CH₂ OH, by the aid of sulfuric acid and peroxidein an aqueous medium and then optionally treating the resultant7-hydroxymethylcamptothecin with an alkylating agent or an acylatingagent to convert the 7-hydroxymethyl group into a 7-alkoxymethyl groupor into a 7-acyloxymethyl group with or without simultaneous acylationof the 20-hydroxy group, or optionally oxidizing the resultant7-hydroxymethylcamptothecin to 7-carboxycamptothecin and then optionallyesterifying the 7-carboxy group with a lower alkanol to form a7-alkoxycarbonyl group, and if desired, converting the free compoundinto an alkali metal salt thereof or vice versa.

Camptothecin used as the starting material may be any of the natural andsynthetically obtained forms, i.e., the d-, l- and dl-forms.

The hydroxymethyl compounds of the general formula [II] are easilycommercially available among which methanol, i.e. the case of A being H,is preferable.

In the first main step, the radical reaction is normally carried out inan aqueous medium in the presence or absence of a transition metal ion.Accordingly, the operation for the first main step is carried out inprinciple by dissolving camptothecin in an aqueous solution of sulfuricacid and a hydroxymethyl compound such as methanol, adding a peroxideand maintaining the mixture under proper reaction conditions until theradical reaction is finished. Any of the peroxides known as radicalreaction initiators can be used as the peroxide in this reaction.Preferable examples of the peroxides include inorganic peroxides such ashydrogen peroxide, persulfuric acid and salts thereof, for example,potassium persulfate, sodium persulfate, ammonium persulfate, bariumperoxide, sodium peroxide, Caro's acid and salts thereof, and calciumperoxide, and organic peroxides such as tert-butyl hydroperoxide,benzoyl peroxide, lauroyl peroxide, caprylyl peroxide, DTBP(di-tert-butyl peroxide) and AIBN (2,2'-azobis-isobutyronitrile). Amongthese organic and inorganic peroxides, the use of hydrogen peroxide, apersulfate such as ammonium peroxide and tert-butyl hydroperoxide ispreferable in the present invention.

The transition metal ion, if it is allowed to be present in the reactionmedium, is supplied to the reaction medium from the corresponding saltwhich is capable of dissociating the ion in the reaction medium.Examples of such transition metal salt include silver salts such assilver nitrate, silver sulfate, silver carbonate and silver acetate,iron salts and oxides such as ferrous sulfate, ferrous chloride and ironmonoxide, copper salts such as cuprous chloride, cupric sulfate, andcupric nitrate, cobalt salt such as cobalt acetate, cobalt sulfate,cobalt nitrate and cobalt acetate, nickel salts such as nickel nitrate,nickel sulfate and nickel chloride, lead salts such as lead acetate,mercury salts such as mercurous chloride and mercuric chloride, andcadmium compounds such as cadmium nitrate and cadmium chloride. Besidesthese compounds, thallium and zinc compounds such as zinc sulfate canalso be used. The use of silver and iron compounds is most preferable.The transition metal ion may not be present in the reaction medium butthe existence of the ion is recommended to promote the radical reactionquickly and efficiently. The transition metal salt is used within therange from an almost equimolar amount to an about 30 molar amount tocamptothecin, preferably in a 10-30 molar amount. If the amount of thetransition metal salt becomes smaller than the equimolar amount tocamptothecin, the effect of promoting the radical reaction will hardlybe recognized. On the other hand, no additional technical merits can beachieved by increasing the amount of the transition metal salt over a 30molar proportion to camptothecin. The use of an excessively large amountof the transition metal salt will rather bring about undesirable effectsin separation of the resulting product from the reaction mixture. Thereaction conditions are represented by temperature and time. Thereaction temperature varies widely from room temperature to the boilingpoint of the reaction mixture. The reaction time is usually withinseveral hours to one day and generally depends on the reactiontemperature adopted. If the transition metal ion is allowed to exist inthe aqueous solution of sulfuric acid and methanol containingcamptothecin and a peroxide is gradually added to the solution, thereaction is promoted at room temperature or in a warmed state to form7-hydroxymethylcamptothecin in a higher yield.

A general operation for performing the first main step comprisesdissolving camptothecin in an aqueous solution of sulfuric acid and thehydroxymethyl compound, adding the radical reaction initiator to theaqueous solution, maintaining the mixture at a temperature within therange from room temperature to the boiling point of the reaction mixturefor several hours to one day and pouring the reaction mixture into icewater to separate the resultant 7-hydroxymethylcamptothecin as aprecipitate from the reaction mixture. The precipitated crude crystalsare collected by filtration or extraction of the reaction mixture with awater-immiscible organic solvent such as methylene chloride, chloroform,ethyl acetate, butyl alcohol, amyl alcohol, carbon tetrachloride andcarbon disulfide. The use of chloroform is preferable for this purpose.The resultant crude 7-hydroxymethylcamptothecin can be purifiedaccording to a usual manner, for example, by recrystallization fromdimethylformamide-dioxane, by thin layer chromatography, by highperformance liquid chromatography or by a combination of thesepurification treatments.

7-Hydroxymethylcamptothecin thus prepared possesses excellentpharmacological properties and can be used directly as a medicament oris useful as an intermediate for preparing various camptothecinderivatives functionally substituted at the hydroxymethyl group in the7-position thereof.

7-Hydroxymethylcamptothecin and 7-substituted camptothecins derivedtherefrom can be converted, if desired, into water-soluble alkali metalsalts by treating the camptothecin derivative in free form with analkali metal hydroxide or carbonate at room temperature or at anelevated temperature. This alkali metal salt is formed by opening of thelactone ring (ring E). However, such alkali metal salt can easily beconverted into the free form by treating the salt with an acid wherebythe lactone ring is formed with simultaneous dehydration(dehydrocyclization). Camptothecin and its derivatives do not form inprinciple acid-addition salts, though they contain two tertiary nitrogenatoms.

The hydroxymethyl group of 7-hydroxymethylcamptothecin can optionally betreated with an alkylating agent to prepare the corresponding7-alkoxymethyl derivatives or with an acylating agent to prepare thecorresponding 7-acyloxymethylcamptothecin or with an oxidizing agent toprepare the corresponding 7-carboxycamptothecin which may further beconverted with a hydroxy compound such as an alcohol into an ester ofcamptothecin-7-carboxylic acid.

The alkylation of 7-hydroxymethylcamptothecin is carried out accordingto a method known per se, for example, by reacting7-hydroxymethylcamptothecin with an alkanol corresponding to the alkylmoiety of 7-alkoxymethylcamptothecin to be prepared. The above reactionis usually carried out by heating the reaction mixture in the presenceof an acid catalyst such as hydrochloric acid, sulfuric acid,fluoroboric acid, benzenesulfuric acid, p-toluenesulfonic acid andborontrifluoride etherate. Illustrative of the alkanol as alkylatingagent are, for example, methanol, ethanol, propanol, butanol, hexanol,deanol and hexadecanol. The use of a lower alkanol such as methanol orethanol is preferable. The operation for this O-alkylation is performednormally by dissolving 7-hydroxymethylcamptothecin in the alkylatingagent, for example, ethanol, adding to the solution any of the acidcatalysts above mentioned and heating the mixture. The resulting7-alkoxymethylcamptothecin can be separated from the reaction mixture byextraction with an organic solvent and purified according to a methodknown per se, for example, by column, thin layer, or high performanceliquid chromatographic techniques or a combination of thesechromatographic techniques.

The acylation of 7-hydroxymethylcamptothecin is carried out according toa method known per se by reacting 7-hydroxymethylcamptothecin with anacylating agent normally in the presence of a dehydration agent or anacid-binding agent. Illustrative of the acylating agent are carboxylicacids and reactive functional derivatives thereof as well as sulfonicacids and reactive functional derivatives thereof and alkylhemisulfates, such as formic acid, acetic acid, propionic acid, butyricacid, phenylacetic acid, succinic acid, trifluoroacetic acid and thelike aliphatic carboxylic acids, benzoic acid and itsnucleus-substituted derivatives, naphthoic acid and the like aromaticacids, alkanesulfonic acids, for example, methanesulfonic acid andethanesulfonic acid, arylsulfonic acids, for example, benzenesulfonicacid, and p-toluenesulfonic acid, and lauryl hemisulfate, and halides orlower alkyl esters of these acids, for example, acetyl chloride andpropionyl bromide. Preferable examples of the acid-binding agent includeinorganic bases such as sodium carbonate, potassium bicarbonate, causticalkali and calcium carbonate, and organic bases such as triethylamine,pyridine and tetramethylammonium hydroxide. In general, the use of areactive functional derivative of the carboxylic or sulfonic acid ispreferable. In case the acylating agent is a carboxylic acid, an acidanhydride, e.g. acetic anhydride is preferably used as the reactivefunctional derivative of the carboxylic acid. The reaction is promotedin the presence of the acid-binding agent at room temperature or at anelevated temperature.

Since 7-hydroxymethylcamptothecin has two hydroxy groups, either of7-acyloxymethylcamptothecins and 7-acyloxy-20-O-acyl-camptothecins areprepared predominantly by properly controlling the proportion of theacylating agent to camptothecin and the reaction temperature. When alarge excess of the acylating agent is used or a higher reactiontemperature is employed, the two hydroxy groups (one of them is aprimary hydroxy group in the hydroxymethyl group and the other is atertiary hydroxy group bound to the 20-position) tend to undergoacylation, thus resulting in the formation of the diacylated product ina larger proportion. Contrary to this, when an almost or slightly excessstoichiometrical amount of the acylating agent is used for the starting7-hydroxymethyl camptothecin or the reaction is carried out at a lowertemperature, for example, at room temperature, the hydroxy group in the7-hydroxymethyl group alone tends to undergo acylation. Accordingly, if7-acyloxymethylcamptothecin are to be prepared predominantly, it isdesirable to use the acylating agent in an almost equimolar amount withrespect to the starting 7-hydroxymethylcamptothecin and to conduct thereaction at a temperature as low as possible. On the other hand, if thediacylated product is to be prepared, the acylating agent should be usedin large excess (at least two molar proportion) and the reaction shouldbe conducted at a higher temperature.

In case a diacylated product wherein the acyl group in the7-acyloxymethyl group is different from that in the 20-position is to beprepared, the acylation of the 7-hydroxymethyl group with an acylatingagent is first carried out carefully under the above mentioned conditionand then the O-acylation of the hydroxy group in the 20-position iscarried out with a different acylating agent.

The acylating reaction is normally performed according to a conventionalmethod by dissolving 7-hydroxymethylcamptothecin in an inert solventcontaining a dehydrating agent or an acid-binding agent and adding anacylating agent while stirring the mixture at room temperature or at anelevated temperature. The reaction can be promoted by using aconventional esterifying catalyst such as sulfuric acid, sodium acetate,pyridine, fluoroboric acid, p-toluenesulfonic acid and strongly acidicion-exchangers such as Amberlite IR-100, IR-105, IR-112 and IR-120 (Rohm& Haas Co., U.S.A.), Dowex 50-X1, X2 and Dowex 30 (Dow Chemical Co.,U.S.A.) and Dia-ion SK #1, K and RK (Mitsubishi Kasei Co., Japan).

7-Hydroxymethylcamptothecin obtained in the first main step can beoxidized in the successive step with an oxidizer capable of convertingthe hydroxymethyl group into a carboxy group according to a method knownper se. Such oxidizer is well known and is selected, for example, fromanhydrous chromates, bichromates and permanganates. This oxidationreaction is usually carried out at room temperature or an elevatedtemperature in the presence of acetone, acetic acid, sulfuric acid orthe like as the reaction medium whereby 7-carboxycamptothecin (orcamptothecin-7-carboxylic acid) is obtained. This carboxylic acid can bepurified, if necessary, by recrystallization from dioxane.

7-Carboxycamptothecin thus obtained may further be converted accordingto a conventional esterification method into7-alkoxycarbonylcamptothecin. This esterification reaction is carriedout in a usual manner by dissolving or suspending 7carboxylcamptothecinin an excess amount of a lower alkanol preferably with 1-8 carbon atoms,adding an esterifying catalyst and heating the mixture. Any of theesterifying catalysts referred to in the acylation of a 7-hydroxymethylgroup is suited for this purpose. Alternatively, a mixture of7-carboxycamptothecin, an at least equimolar amount of a lower alkanoland an esterifying catalyst in benzene to toluene is refluxed whileremoving only water formed during the reaction and distilled as anazeotrope with the solvents by means of an appropriate water-separator.Illustrative of the lower alkanol are, for example, methanol, ethanol,n-propanol, isopropanol, n-butanol, tert-butanol, hexanol, heptanol andoctanol. Preferable examples of the esterifying catalyst are well knownin the art and include sulfuric acid, hydrochloric acid,p-toluenesulfonic acid and borontrifluoride etherate. The resultingester can be purified by recrystallization, for example, fromethanol-dioxane.

According to a variant of the first main step of this embodiment,camptothecin derivatives of the general formula: ##STR4## wherein X" isan alkyl group or an aralkyl group, are prepared by subjectingcamptothecin to a radical reaction with an organic compound of thegeneral formula:

    X"--Q                                                      [II']

wherein Q is the grouping --CH₂ OH, --COOH, --CHO, --COX" or ##STR5##and X" has the same meaning as given above, by the aid of sulfuric acidand a peroxide in an aqueous medium in the presence of a transitionmetal ion.

When Q is --CH₂ OH, the organic compound of the general formula [II'] isan alkanol or an aralkanol. Preferable examples of such alkanol oraralkanol include straight or branched chain primary alcohols such asethanol, propanol, butanol, pentanol, hexanol, octanol,3-methylpentanol, isoamyl alcohol, cyclohexylmethanol,cyclopentylmethanol, decanol, phenethyl alcohol and phenylpropanol. WhenQ is --COOH, the organic compound is a fatty acid or an arylfatty acids.Illustrative of such acid are, for example, acetic acid, propionic acid,butyric acid, hexanoic acid, octanoic acid, phenylacetic acid,β-phenylpropionic acid. When Q is --CHO, the organic compound is analdehyde such as acetaldehyde, propionaldehyde, butyraldehyde,caprylyldehyde or phenylacetaldehyde. When Q is --COX", the organiccompound is a dialkyl ether or a diaralkyl ether. In this case, the twoalkyl moieties of the dialkyl ether may be the same or different.Similarly, when Q is ##STR6## the organic compound is for example, atert-alkyl hydroperoxide in which the three alkyl groups may be the sameor different. A preferable compound of this type is tert-butylhydroperoxide.

This radical reaction is carried out in principle by dissolving in watera transition metal salt capable of dissociating the transition metalion, camptothecin, sulfuric acid and a compound of the general formula[II'] in any order of succession and adding a peroxide and stirring themixture. Usually, a compound of the general formula [II'] and atransition metal salt are dissolved in water and then camptothecin andsulfuric acid are added in the noted order to the solution. A peroxideis added to the solution under agitation and ice cooling and theagitation is continued even after the temperature is raised to roomtemperature. After completion of the reaction, ice water is added to thereaction mixture and the resulting product is extracted with awater-immiscible organic solvent such as chloroform and purified, forexample, by column chromatography followed by recrystallization from anorganic solvent such as n-hexane-chloroform.

Preferable examples of the transition metal salt capable of dissociatingthe transition metal ion in the reaction medium include silver saltssuch as silver nitrate, silver sulfate, silver carbonate and silveracetate, iron salts and oxides such as ferrous sulfate, ferrous chlorideand iron monoxide, copper salts such as cuprous chloride, cupricsulfate, cupric nitrate, cobalt salts such as cobalt chloride, cobaltsulfate, cobalt nitrate and cobalt acetate, nickel salts such as nickelnitrate, nickel sulfate and nickel bromide. Besides these compounds,salts of lead, mercury, cadmium thallium and zinc, such as lead acetate,mercurous chloride, cadmium nitrate and zinc sulfate, can also be usedequivalently. Examples of the peroxide include inorganic peroxides suchas hydrogen peroxide, persulfates such as potassium persulfate, sodiumpersulfate, Caro's acid and its salts, barium peroxide, calciumperoxide, sodium peroxide and organic peroxides such as tert-butylhydroperoxide, benzoyl peroxide, lauroyl peroxide, caprylyl peroxide,DTBP and AIBN.

In case a higher alcohol or the like compound which is sparingly solublein water is used as a compound of the general formula [II'], adissolution assistant is used to promote dissolution of such sparinglysoluble compound in water. Utilizable as the dissolution assistant arepolar organic solvents which are inert to the reaction and capable offorming a homogeneous phase, such as acetic acid, dimethylformamide,acetonitrile, dioxane, dimethoxyethane and tetrahydrofuran. Varioussurfactants capable of forming a homogeneous phase, particularlynonionic surfactants may also be used for this purpose in place of thedissolution assistant.

The compound of the general formula [II'] is preferably used in a largeexcess in molar ratio to camptothecin. For example, about 20 molarproportion of the compound is used for camptothecin. The transitionmetal salt and the peroxide are used respectively in excess, forexample, about 5-8 molar excess to the amount of camptothecin used. Thereason why the compound of the general formula [II'] is used in largeexcess is that an excessively sufficient amount of a radical speciesallowed to be present in the reaction medium serves not only to preventoccurrence of any side reaction which affords by-products but also topromote the formation of the end product normally within a reasonableperiod of time.

In the above radical reaction, it is of interest that when a straight orbranched chain primary alcohol such as ethanol or isobutanol is used asthe compound of the general formula [II'], the moiety of X", e.g. methylgroup in the case of using ethanol or isopropyl group in the case ofusing isobutanol (i.e. the moiety of the primary alcohol excluding theterminal grouping --CH₂ OH) is introduced into the 7-position ofcamptothecin. In other words, an alkyl moiety of the alcohol from whichone carbon atom has been excluded is introduced in all cases into the7-position of camptothecin. When a cycloalkylmethanol such ascyclohexylmethanol or cyclopentylmethanol is used as the organiccompound X"--Q, the cycloalkyl group can directly be introduced into the7-position of camptothecin. In a similar manner, an aralkyl group suchas a benzyl group can be introduced by using an aralkanol composed ofthe aralkyl moiety and the grouping CH₂ OH, e.g. phenethyl alcohol asthe organic compound X"--Q. When a fatty acid or an arylfatty acid isused as the organic compound X"-- Q, the moiety of the carboxylic acidfrom which the grouping --COOH has been eliminated is introduced intothe 7-position of camptothecin. For example, when acetic acid orisovaleric acid is used as the organic compound, a methyl group orisobutyl group is introduced into the 7-position of camptothecin,respectively. Accordingly, the use of phenylacetic acid serves tointroduce a benzyl group into camptothecin. The same applies to the casewherein an aldehyde of X"--CHO is used as the organic compound. In thiscase, the moiety of such aldehyde from which the grouping CHO has beenremoved is introduced into the 7-position of camptothecin. Thus, the useof acetaldehyde or propionaldehyde affords 7-methyl- or7-ethylcamptothecin, respectively. When a ketone X"--CO--X" (Q═COX") inwhich two alkyl and/or aralkyl moieties (X") may be the same ordifferent is used as the organic compound X"--Q, either of the moieties(X") is introduced into the 7-position of camptothecin. If a symmetricalketone such as acetone or diethyl ketone is used, a methyl group orethyl group is introduced. If, however, an asymmetrical ketone such asmethyl ethyl ketone or methyl isobutyl ketone is used as the organiccompound, a mixture of a methyl group and ethyl group or a mixture of amethyl group and isobutyl group is introduced into the 7-position ofcamptothecin. In case the organic compound X"--Q is a hydroperoxide, Qis the grouping ##STR7## In case tert-butyl hydroperoxide is used as theorganic compound X"--Q, the three alkyl moiety (X") is the same and amethyl group is introduced into the 7-position of camptothecin. However,when one or two alkyl moieties (X") in Q are different, a mixture of twoor three different kinds of alkyl group is introduced into the7-position of camptothecin. Thus, care should be taken when the organiccompound X"--Q is a ketone or hydroperoxide.

7-Alkylcamptothecins obtained in any of the above mentioned variant ofthe first step can optionally be treated with an oxidizing agent to formthe corresponding 7-carboxycamptothecin (camptothecin-7-carboxylic acid)which may further be esterified, if desired, with a lower alkanol toform an ester thereof (a lower alkyl camptothecin-7-carboxylate).

Any of the oxidizers capable of oxidizing an alkyl group such as amethyl group bound to an aromatic ring to a carboxyl group can be usedfor this optional oxidation treatment. Such oxidizer is well known inthis art and is usually selected, for example, from anhydrous chromates,bichromates and permanganates.

This oxidation reaction is usually carried out at room temperature or atan elevated temperature in the presence of acetone, acetic acid,sulfuric acid or the like reaction medium. 7-Carboxycamptothecin thusobtained can be purified, if necessary, by recrystallization fromdioxane. This product (7-carboxycamptothecin) may further be converted,if desired, according to a conventional esterification method [asdescribed in the case of obtaining a lower alkylcamptothecin-7-carboxylate from 7-hydroxymethylcamptothecin via7-carboxycamptothecin (camptothecin-7-carboxylic acid) in the optionalafter-treatment in this embodiment] into an ester ofcamptothecin-7-carboxylate.

According to another embodiment of the process of the present invention,camptothecin derivatives of the general formula: ##STR8## wherein Y' isOH or the grouping OR³ where R³ is a lower alkyl group or an acyl groupand Z is H or an acyl group, as well as water-soluble alkali metal saltsare prepared by treating camptothecin with sulfuric acid and apersulfate in an aqueous medium containing a transition metal ion, andthereafter optionally treating the resultant 5-hydroxycamptothecin withan alkylating agent or an acylating agent to convert the hydroxy groupin the 5-position into a 5-alkoxy group or into a 5-acyloxy group withor without simultaneous acylation of the hydroxy group in the20-position.

In the first main step, the radical reaction is in principle carried outby dissolving camptothecin in a mixture of sulfuric acid and water,adding a transition metal salt and an aqueous solution of a persulfateto the solution and stirring the mixture while heating. Preferableexamples of the transition metal salt capable of dissociating thetransition metal ion in the reaction medium include silver salts such assilver nitrate, silver sulfate, silver carbonate and silver acetate,iron salts and oxides such as ferrous sulfate, ferrous chloride and ironmonoxide, copper salts such as cuprous chloride, cupric sulfate andcupric nitrate, cobalt salts such as cobalt chloride, cobalt sulfate,cobalt nitrate and cobalt acetate, nickel salts such as nickel sulfateand nickel nitrate. Salts of lead, mercury, thallium, cadmium and zincsuch as lead acetate, mercuric chloride, cadmium nitrate and zincsulfate can also be used equivalently. Preferable are silver salts andiron compounds as mentioned above.

Illustrative of the persulfate are, for example, sodium persulfate,potassium persulfate, ammonium persulfate, and Caro's acid and itssalts. In addition to these persulfates, any of the peroxides which arecapable of forming a persulfate in the reaction medium containingsulfuric acid can also be used equivalently to the inherent persulfate.Preferable examples of such persulfate-forming peroxide are, forexample, hydrogen peroxide, barium peroxide, calcium peroxide, sodiumperoxide and organic peroxides such as tert-butyl hydroperoxide, benzoylperoxide, lauroyl peroxide, caprylyl peroxide, DTBP and AIBN. Thepersulfate or a peroxide capable of forming a persulfate is used in anamount within the range of 5-30 molar proportion with respect tocamptothecin. The mixing ratio of sulfuric acid to water is within therange from 10:90 to 90:10, with the ratio of 50:50 being preferable. Nocritical limitation exists in the proportion of camptothecin to sulfuricacid, but sulfuric acid is usually used in a large excess. Thetransition metal salt is normally used in an equimolar amount withrespect to the amount of camptothecin used, but the salt may be used inexcess. The reaction mixture can be heated up to the boiling temperaturethereof and the reaction is usually finished within several hours afteraddition of the persulfate.

The radical reaction may be effected in the presence of acetic acid,bromoacetic acid, glycolic acid, dimethylformamide or the like polarsolvent whereby the reaction time can be shortened and the yield of theproduct can be increased.

Diastereomers of 5-hydroxycamptothecin exist in connection with theconfiguration of the hydroxy group in the 5-position thereof. The twodiastereomers can be separated by converting 5-hydroxycamptothecin into5-acetoxycamptothecin and subjecting the latter to chromatography withsilica gel. More precisely, 5-acetoxycamptothecin (a mixture of the twodiastereomers) is subjected to thin layer chromatography with 1%methanol-chloroform as developing solvent whereby the5-acetoxycamptothecin can be separated into the individual isomershaving Rf values of 0.20 and 0.15. According to the NMR spectrograph ofthe two isomers, the methine proton in the 5-position and the methylmoiety of the acetoxy group in the 5-position of both isomers areobserved at δ 7.96; 2.192 ppm and δ 7.91; 2.195 ppm, respectively.However, no substantial difference is observed between both isomers inpeaks based on other hydrogens. In an NMR spectrograph of5-hydroxycamptothecin prepared in the first main step, the methineproton in the 5-position is observed as two singlet peaks at δ 6.66 andδ 6.72 ppm each by 0.5 H. Thus, it is confirmed that5-hydroxycamptothecin is a mixture (about 1:1) of the two diastereomers.

The new 5-hydroxycamptothecin thus prepared possesses excellentpharmacological properties and can be used directly as a medicament oris useful as an intermediate product for preparing various camptothecinderivatives functionally substituted at the hydroxy group in the5-position thereof.

The hydroxy group in the 5-position of the 5-hydroxycamptothecin canoptionally be treated with an alkylating agent to prepare thecorresponding 5-alkoxycamptothecin derivatives or with an acylatingagent to prepare the corresponding 5-acyloxycamptothecin derivatives.

The alkylation of 5-hydroxycamptothecin is conducted according to amethod known per se, for example, by reacting 5-hydroxycamptothecin witha lower alkanol corresponding to the alkyl moiety R³ of5-alkoxycamptothecin to be prepared. The above reaction is usuallycarried out by heating the reaction mixture in the presence of an acidcatalyst such as hydrochloric acid, sulfuric acid, fluoroboric acid,benzenesulfonic acid, p-toluenesulfonic acid and boron trifluorideetherate. Illustrative of the lower alkanol as alkylating agent are, forexample, methanol, ethanol, n-propanol, isopropanol, n-butanol,isobutanol, tert-butanol, n-pentanol, n-hexanol, heptanol and octanol.The use of methanol or ethanol is preferable in ease of availability andhandling. The operation for this O-alkylation is performed normally bydissolving 5-hydroxycamptothecin in an excess amount of the alkylatingagent, for example, ethanol, adding to the solution any of the abovementioned catalysts and heating the mixture. The resulting5-alkoxycamptothecin can be separated from the reaction mixture byextraction with an organic solvent and purified according to a methodknown per se, for example, by column or thin layer chromatographythrough silica gel.

The acylation of 5-hydroxcamptothecin is carried out according to amethod known per se by reacting 5-hydroxycamptothecin with an acylatingagent preferably in the form of a reaction derivative of an acid, ifnecessary, in the presence of an acid-binding agent. Examples of theacylating agent include carboxylic acids and reactive functionalderivatives thereof as well as sulfonic acids and reactive functionalderivatives thereof and alkyl hemisulfates. Illustrative of suchacylating agent are formic acid, acetic acid, propionic acid, butyricacid, phenylacetic acid, succinic acid, trifluoroacetic acid, and thelike aliphatic carboxylic acids, benzoic acid and itsnucleus-substituted derivatives, naphthoic acid and the like aromaticacids, alkanesulfonic acids, for example, methanesulfonic acid andethanesulfonic acid, arylsulfonic acids, for example, benzenesulfonicacid and p-toluenesulfonic acid, and lauryl hemisulfate, and halides orlower alkyl esters of these acids, for example, acetyl chloride andpropionyl bromide. In case the acylating agent is a carboxylic acid, anacid anhydride is preferably used as a reactive functional derivative ofthe carboxylic acid. Preferable examples of the acid-binding agent whichis used to promote the reaction between 5-hydroxycamptothecin and theacylating agent in the form of a reactive functional derivative of anacid include inorganic bases such as sodium carbonate, potassiumbicarbonate, caustic alkali and calcium carbonate, and organic basessuch as triethylamine, pyridine, picoline, lutidine, collidine andtetramethylammonium hydroxide. In general, the use of a reactivefunctional derivative of the carboxylic or sulfonic acid is preferableand the reaction is promoted in the presence of the acid-binding agentat room temperature or at an elevated temperature.

Since 5-hydroxycamptothecin has two hydroxy groups, either of5-acyloxycamptothecins and 5-acyloxy-20-O-acyl-camptothecins areprepared mainly by properly controlling the proportion of the acylatingagent and the reaction temperature. When a large excess of the acylatingagent is used or a higher reaction temperature is employed, the twohydroxy groups tend to undergo acylation, thus resulting in theformation of the diacylated product in a larger proportion. Contrary tothis, when an almost or slightly excess stoichiometrical amount of theacylating agent is used for the 5-hydroxycamptothecin or the reaction iscarried out at a lower temperature, for example, at room temperature,the hydroxy group in the 5-position alone tends to undergo acylation.Accordingly, if 5-acyloxycamptothecins are to be prepared chiefly, it isdesired to use the acylating agent in an almost equimolar amount withrespect to the 5-hydroxycamptothecin and to conduct the reaction at atemperature as low as possible. On the other hand, if the diacylatedproduct is to be prepared, the acylating agent must be used in largeexcess (at least two molar proportion) and the reaction should beconducted at a higher temperature.

Although two diastereomers (R- and S-isomers) of 5-hydroxycamtothecinexist in connection with the configuration of the hydroxy group in the5-position thereof, they can be separated by converting the 5-hydroxycompound into the 5-acetoxy compound and subjecting the latter to thinlayer chromatography in a manner as described above.

5-Hydroxycamptothecin and 5-substituted camptothecins derived therefromcan be converted, if desired, into water-soluble alkali metal salts bytreating the camptothecin derivative in free form with an alkali metalhydroxide or carbonate at room temperature or at an elevatedtemperature. This alkali metal salt is formed by opening of the lactonering (ring E). However, such alkali metal salt can easily be convertedinto the free form by treating the salt with an acid whereby the lactonering is formed with simultaneous dehydration (dehydroxyclization).

The new camptothecin derivatives of the present invention exhibit atleast one of a high level of anti-tumor activity and a very slighttoxicity. As a result of animal tests, it has been found that themajority of the camptothecin derivatives of the present invention aresuperior to camptothecin itself in anti-tumor activity to lymphaticleukemia L-1210 (from the National Cancer Institute strain). Thetoxicity of the new camptothecin derivatives of the present invention isgenerally low. The pharmacological activity of the new camptothecinderivatives of the present invention is as high as 100-380% in terms ofT/C % (median life-span of treated mice divided by median life-span ofuntreated control mice in percentage--one of the standard methods forevaluating anti-tumor activity) in comparison with camptothecin itself.Thus, the new camptothecin derivatives of the present invention areuseful as anti-tumor agents or as intermediate products for preparingother useful derivatives.

The present invention will now be illustrated in more detail by way ofexamples. In these examples, the relation between part and percentage isby weight unless otherwise indicated.

All melting points were measured in a capillary tube and wereuncorrected. NMR spectra were obtained by Hitachi R-22 90MC and JEOLFX-100 NMR spectrophotometer, using TMS as an internal reference. Massspectra were recorded on a Hitachi RMS-4 or a JEOL JMS D300 instrument.The IR and UV spectra were measured on a JASCO IRA-1 or on a HitachiEPS-3 spectrophotometer, respectively. Optical rotation was measured ona Yanagimoto Yanaco OR-50 automatic polarimeter.

EXAMPLE 1 (Preparation of 7-hydroxymethylcamptothecin)

Camptothecin (100 mg, 0.287 m-mol) was suspended in methanol (25 ml) andthen dissolved therein by addition under ice-cooling of 75% sulfuricacid (10 ml). To the solution was added dropwise under reflux withstirring an aqueous solution (100 ml) of ammonium persulfate (15 g,0.0657 mol) over 16 hours. The reaction mixture was poured into icewater (100 ml) and the organic matter was extracted with a mixture (1:1,500 ml) of dioxane-chloroform and then thrice with chloroform (100ml×3). The organic phases were combined, dried with anhydrous magnesiumsulfate, filtered and then evaporated until dryness under reducedpressure. The remaining solid having an orange color was warmed (50°-60°C.) with methanol (200 ml) and stirred for 30 minutes. An insolublematter was collected by filtration, dried under reduced pressure andrecrystallized from dimethylformamide-dioxane whereupon 40 mg (36.9%) of7-hydroxymethylcamptothecin were obtained as light yellow whiteprismatic crystals having a melting point of 274°- 276° C. (dec.). Rfvalue 0.125 (5% methanol-chloroform).

IRν_(max) ^(KBr) cm⁻¹ : 3300, 2960, 1770, 1665, 1605, 1470, 1200, 1170,1115, 770.

NMR (DMSO-d₆) δ ppm: 0.90(3H, t, J=7 Hz), 1.88(2H, q, J=7 Hz), 5.23(2H,s) 5.34(2H, s), 5.40(2H, s), 7.30(1H, s), 7.55-8.13(4H, m).

MS m/e: 378.1283 [M⁺ ] (C₂₁ H₁₈ N₂ O₅ =378.1209).

UV λ_(max) ^(EtOH) nm: 220, 245, 253.5, 292, 302, 335(sh), 359, 372.

EXAMPLE 2 (Preparation of 7-hydroxymethylcamptothecin)

Camptothecin (3.00 g, 8.61 m-mol) was suspended in methanol (90 ml) andthen dissolved therein by addition of 75% sulfuric acid (75 ml) and 75ml of water. To the solution was added FeSO₄. 7H₂ O (40 g, 0.143 mol)and then was added dropwise under ice-cooling and agitation 30% hydrogenperoxide (15 ml) over 2 hours. After addition of the hydrogen peroxide,the reaction mixture was stirred for 14 hours at room temperature andpoured into ice water (1 l). The yellowish brown solid precipitated wascollected by filtration and dried under reduced pressure whereby 2.5 gof 7-hydroxymethylcamptothecin were obtained. The filtrate was extractedwith chloroform (250 ml×4) thereby obtaining 200 mg of crude crystals ofthe product. 2.7 Grams (82.9%) in toto of 7-hydroxymethylcamptothecinwere obtained, which was identified to be the same product as obtainedin Example 1 by way of IR-absorption spectra and thin layerchromatography.

EXAMPLE 3 (Preparation of 7-hydroxymethylcamptothecin)

Camptothecin (50 mg, 0.143 m-mol) was dissolved in 75% sulfuric acid (3ml). To this solution were added glycolic acid (500 mg, 6.57 m-mol) andsilver nitrate (250 mg, 1.31 m-mol) and then was added dropwise underheating (100°-110° C.) and agitation and aqueous solution (15 ml) ofammonium persulfate (3.00 g, 0.0131 m-mol) over 2 hours. After thereaction mixture was allowed to cool, ice water (100 ml) was poured intothe reaction mixture which was then extracted with chloroform (100ml×3). The chloroform layers were combined, washed with a 7% aqueoussolution of sodium bicarbonate (300 ml) and then with a saturated ediblesalt solution (100 ml), dried with anhydrous magnesium sulfate, filteredand evaporated until dryness under reduced pressure. The residue wassubjected to thin layer chromatography (5% methanol-chloroform) toeffect separation and purification of 7-hydroxymethylcamptothecinwhereupon 10.3 mg (19.0%) of pure 7-hydroxymethylcamptothecin wereobtained. Besides this, 10.7 mg of camptothecin were recovered.

EXAMPLE 4 (Preparation of 5-hydroxycamptothecin)

Camptothecin (100 mg, 0.287 m-mol) was dissolved in 75% sulfuric acid (5ml). To this solution was added silver nitrate (50 mg, 0.295 m-mol) andthen was added dropwise under heating (100°-110° C.) and agitation anaqueous solution (20 ml) of ammonium persulfate (1.96 g, 8.59 m-mol)over 1.5 hours. The heating and agitation were continued for 3 hours.After the reaction mixture was allowed to cool, the reaction mixture wasdiluted with water to 300 ml and was then extracted with chloroform (100ml×5). The chloroform layers were combined, dried with anhydrousmagnesium sulfate, filtered and evaporated until dryness under reducedpressure. The residue was subjected to thin layer chromatography toeffect separation and purification whereupon 10.5 mg of camptothecinwere recovered and 5-hydroxycamptothecin was obtained as a light yellowwhite solid. The yield was 10.3 mg (10.8%).

IRν_(max) ^(KBr) cm⁻¹ : 3340, 2960, 1750, 1660, 1600, 1235, 1165.

NMR (DMSO-d₆) δ ppm: 1.01(3H, t, J=7.5 Hz), 1.91(2H, q, J=7.5 Hz),4.87(1H, br, D₂ O ex.), 5.25, 5.52(two 1H s, dxd, J=17 Hz), 6.66(0.5H,s), 6.72(0.5H, s, 7.31(1H, br, s), 7.50-8.35(4H, m), 8.52(1H, br, s).

MS m/e: 364.1041 [M⁺ ] (C₂₀ H₁₆ N₂ O₅ =364.1053).

UV λ_(max) ^(EtOH) nm: 217, 224(sh), 248(sh), 257, 295, 336(sh), 357,370(sh).

EXAMPLE 5 (Preparation of 5-hydroxycamptothecin)

Camptothecin (350 mg, 1 m-mol) was dissolved in concentrated sulfuricacid and bromoacetic acid (13.9 g) while warming. To this solution wasadded silver nitrate (170 mg, 1 m-mol) and then was added dropwise underheating (110°-120° C.) and agitation an aqueous solution (100 ml) ofammonium persulfate (7.0 g, 0.0307 mol) over about 3 hours. The heatingand agitation were continued for one hour. After the reaction mixturewas allowed to cool, the reaction mixture was diluted with water to 100ml and was then extracted with chloroform-dioxane(500 ml-200 ml). Theorganic phase was washed with a 5% aqueous solution of sodiumbicarbonate (500 ml), dried with anhydrous magnesium sulfate, filteredand evaporated until dryness under reduced pressure. The residue waspurified by recrystallization from n-hexane-chloroform whereupon5-hydroxycamptothecin was obtained as a light yellow white solid. Theyield was 149 mg (39%). This product was identified to be the same asthe sample obtained in Example 4 by way of IR-absorption spectra andthin layer chromatography.

EXAMPLE 6 (Preparation of 5-hydroxycamptothecin)

Camptothecin (1.30 g, 3.69 m-mol) was dissolved in 45% sulfuric acid (40ml). To this solution were added bromoacetic acid (1.54 g), 0.011 mol)and ferrous sulfate heptahydrate (1.02 g, 3.69 m-mol) and then was addeddropwise under heating (90°-100° C.) and agitation an aqueous solution(100 ml) of ammonium persulfate (3.80 g, 0.016 mol) in portions over 4.5hours. The heating and agitation were continued for 3.5 hours. After thereaction mixture was allowed to cool, the reaction mixture was dilutedwith ice water to about one liter and was then extracted with chloroform(250 ml×6). The chloroform layers were combined, dried with anhydrousmagnesium sulfate, filtered and evaporated until dryness under reducedpressure. The residue was recrystallized from n-hexane-chloroformwhereupon 5-hydroxycamptothecin was obtained as a light yellow whitesolid. The yield was 656 mg (48.5%). This product was identical inIR-absorption spectra with the product obtained in the precedingExample.

EXAMPLE 7 (Preparation of 20-O-acetyl-7-acetoxymethylcamptothecin)

7-Hydroxymethylcamptothecin (30 mg, 0.0793 m-mol) was dissolved inacetic anhydride (1 ml). Pyridine (0.1 ml) was added to the solution andthe mixture was heated (110°-120° C.) with stirring for 2 hours. Thereaction mixture was evaporated until dryness under reduced pressure.Water (3 ml) was added to the residue and the precipitate was collectedby filtration, thoroughly washed with water (5 ml) and dried underreduced pressure whereupon crude crystals of 20-O-acetyl-7-acetoxymethylcamptothecin were obtained. The yield was 35 mg (98.5%).Recrystallization of this crude product from ethyl alcohol forpurification gave 25 mg (68.4%) of light yellow white needle crystals.M.P. 291°-293° C. (dec.).

IRν_(max) ^(KBr) cm⁻¹ : 2980, 1750, 1670, 1610, 1240, 770.

NMR (CDCl₃) δ ppm: 0.99(3H, t, J=7.5 Hz), 2.24(2H, q, J=7.5 Hz),2.19(3H, s), 2.23(3H, s), 5.47(2H, s), 5.57, 5.75(two 1H s, dxd, J=18Hz), 5.77(2H, s), 7.26(1H, s), 7.63-8.46(4H, m).

MS m/e: 462.1401 [M⁺ ] for C₂₅ H₂₂ N₂ O₇ =462.1419.

EXAMPLE 8 (Preparation of 7-acetoxymethylcamptothecin)

7-Hydroxymethylcamptothecin (150 mg, 0.431 m-mol) was dissolved in amixture of pyridine (20 ml) and dimethylformamide (2 ml). To thissolution was added under agitation at room temperature acetic anhydride(105 mg, 1.03 m-mol) in small portions over 7 hours. The reactionmixture was evaporated until dryness under reduced pressure and theresidue was taken up in chloroform (250 ml). The solution was shakenfirst with a 5% aqueous solution (100 ml) of sodium bicarbonate and thenwith 5% hydrochloric acid (100 ml). The chloroform layer obtained waswashed with a saturated edible salt solution (100 ml), dried withanhydrous magnesium sulfate, filtered and evaporated until dryness underreduced pressure. The residue was purified by recrystallization fromn-hexane-chloroform whereupon 142 mg (78.4%) of7-acetoxymethylcamptothecin were obtained as light yellow white needlecrystals. M.P. 277°-279° C. (dec.).

IRν_(max) ^(KBr) cm⁻¹ : 3320, 2970, 1770, 1660, 1610, 1235, 770.

NMR (in CDCl₃) δ ppm: 1.03(3H, t, J=7 Hz), 1.89(2H, q, J=7 Hz), 2.18(3H,s), 5.44(2H, s), 5.26, 5.72(two 1H s, dxd, J=16.5 Hz), 5.71(2H, s), 7.70(1H, s), 7.58-8.28 (4H, m).

MS m/e: 420.1369 [M⁺ ] for C₂₃ H₂₀ N₂ O₆ =420.1314.

UVλ_(max) ^(EtOH) nm: 220, 246(sh), 255, 292, 336(sh), 360, 373.

EXAMPLE 9 (Preparation of 7-succinoyloxymethylcamptothecin)

7-Hydroxymethylcamptothecin (200 mg, 0.529 m-mol) was dissolved inpyridine (30 ml). To this solution were added under heating (70°-80° C.)and agitation 100 mg of solid succinic anhydride as such. The heatingand agitation were continued for 12 hours. The mixture was reactedtogether for 3 days under continuous heating and agitation whilesupplying additional succinic anhydride in an amount of 100 mg per day.Thus, 400 mg (4 m-mols) of succinic anhydride in all were used for thereaction. The resultant reaction mixture was evaporated until drynessunder reduced pressure and the residue was taken up in ethanol anddecolored with active carbon. The ethanol was evaporated until drynessunder reduced pressure whereupon 235 mg (93%) of light yellow whiteprismatic crystals were obtained. This crude product was purified byrecrystallization from n-hexane-ethanol whereupon 185 mg (73.2%) of7-succinoyloxymethylcamptothecin were obtained as light yellow whiteprismatic crystals. M.P. 287-289 (dec.).

IRν_(max) ^(KBr) cm⁻¹ : 3400-2750, 2960, 1750(vs), 1660, 1605, 1420,1170, 775.

NMR (DMSO-d₆) δ ppm: 0.94(3H, t, J=7 Hz), 1.94(2H, q, J=7 Hz), 2.55(4H,br), 5.44(4H, brs), 5.80(2H, s), 7.40(1H, s), 7.65-8.43(4H, m).

EXAMPLE 10 (Preparation of20-O-trifluoroacetyl-7-trifluoroacetoxymethylcamptothecin)

7-Hydroxymethylcamptothecin (200 mg, 0.529 m-mol) was dissolved inpyridine (40 ml) under warming. After allowing the solution to cool,trifluroacetic anhydride (300 mg, 1.43 m-mol) was added and the mixturewas stirred for 8 hours at 40° C. The reaction mixture was concentrateduntil dryness under reduced pressure and the residue was subjected toseparation and purification by way of column chromatography (chloroform)through silica gel (50 g) whereby20-O-trifluoroacetyl-7-trifluoroacetoxymethylcamptothecin was obtainedas light yellow white crystals. The yield was 120 mg (39.7%).

IRν_(max) ^(KBr) cm⁻¹ : 3340, 1760, 1660, 1600, 1235, 1160, 765.

M.P. 264°-267° C. (dec.).

EXAMPLE 11 (Preparation of 7-benzoyloxymethylcamptothecin)

7-Hydroxymethylcamptothecin (200 mg, 0.592 m-mol) was dissolved inpyridine (30 ml) under warming. Benzoyl chloride (260.2 mg, 1.85 m-mol)was added to this solution and the mixture was stirred for 15 hours at50°-60° C. The pyridine was distilled off under reduced pressure and theresidue was taken up in chloroform (30 ml). The solution was shakenfirst with a 5% aqueous solution (500 ml) of sodium hydrogen carbonateand then with 5% hydrochloric acid (500 ml). The chloroform layer wasdried with anhydrous magnesium sulfate, filtered and evaporated untildryness under reduced pressure. The residue was purified byrecrystallization from methanol whereupon 148 mg (57.9%) of7-benzoyloxymethylcamptothecin were obtained as light yellow whiteneedle crystals. M.P. 298° C.˜(dec.).

IRν_(max) ^(KBr) cm⁻¹ : 3420, 2940, 1770, 1730, 1675, 1610, 1460, 1280,1110, 770, 715.

NMR (CDCl₃) δ ppm: 1.09(3H, t, J=7.5 Hz), 1.93(2H, q, J=7.5 Hz),5.55(4H, br s), 5.92(2H, s), 7.47(1H, s), 7.54-8.30(9H, m).

MS m/e: 482 [M⁺ ] (C₂₈ H₂₂ N₂ O₆ =482).

EXAMPLE 12 (Preparation of 7-propionyloxymethylcamptothecin)

7-Hydroxymethylcamptothecin (378 mg, 1 m-mol) was dissolved in anhydrousdimethylformamide (80 ml) under warming. After cooling of the solution,anhydrous pyridine (1 ml) and propionic anhydride (1 ml, 8 eq.) wereadded and the mixture was stirred for 24 hours at room temperature.After completion of the reaction, ethanol (10 ml) was added to themixture with stirring for a while to decompose the excess of theanhydride and the solvents were then distilled off under reducedpressure. The residue was purified by way of column chromatographythrough silica gel (10 g) whereby 420 mg (96.8%) of crude7-propionyloxymethylcamptothecin crystals were obtained.Recrystallization of the crude product from n-hexane-chloroform gave 210mg (48.4%) of light yellow white needle crystals. M.P. 279°-280° C.

IR_(max) ^(KBr) νcm⁻¹ : 3230, 1740, 1655, 1595, 1460, 1165, 765.

NMR (in CDCl₃) δ ppm: 1.05(3H, t, J=7 Hz), 1.18(3H, t, J=7 Hz), 1.91(2H,q, J=7 Hz), 2.47(2H, q, J=7 Hz), 5.28(2H, d, J=7 Hz), 5.44(2H, s),5.70(2H, d, J=17 Hz), 5.71(2H, s), 7.63(1H, s) 7.70-8.30(4H, m).

MS: m/e 434 [M⁺ ] for C₂₄ H₂₂ N₂ O₆ =434.15.

EXAMPLE 13 (Preparation of 7-butyryloxymethylcamptothecin)

7-Hydroxymethylcamptothecin (378 mg, 1 m-mol) was dissolved in anhydrousdimethylformamide (80 ml) under warming. Anhydrous pyridine (1 ml) wasn-butyric anhydride (1 ml, about 8 eq.) were added to this solution andthe mixture was stirred for 4 hours at 60° C. After completion of thereaction, 10 ml of ethanol were added to the reaction mixture withstirring for a while to effect decomposition of the excess of theanhydride and the solvents were then removed by distillation underreduced pressure. The residue was purified by way of columnchromatography (chloroform) through silica gel (10 g) followed byrecrystallization from n-hexane-chloroform whereby 160 mg (35.7%) of7-butyryloxymethylcamptothecin were obtained as light yellow whiteneedle crystals. M.P. 252°-2.54° C.

IR_(max) ^(KBr) νcm⁻¹ : 3350, 1750, 1740, 1665, 1600, 1435, 1155, 770.

NMR (in CDCl₃) δ ppm: 0.94(3H, t, J=7 Hz), 1.05(3H, t, J=7 Hz), 1.69(2H,sex, J=7 Hz), 1.91(2H, q, J=7 Hz), 2.42(2H, t, J=7 Hz), 5.28(2H, d, J=16Hz), 5.44(2H, s), 5.69(2H, s), 5.72(2H, d, J=16 Hz), 7.63(1H, s),7.58-8.25(4H, m).

MS: m/e 448 [M⁺ ] for C₂₅ H₂₄ N₂ O₆ =448.16.

EXAMPLE 14 (Preparation of 7-octanoyloxymethylcamptothecin)

7-Hydroxymethylcamptothecin (200 mg, 0.53 m-mol) was dissolved in warmedanhydrous pyridine (50 ml). n-Octanoyl chloride (260 mg, 3 eq) was addedto this solution and the mixture was stirred for 2 hours at 80° C. Thesolvent was then distilled off under reduced pressure and the residuewas taken up in chloroform (100 ml). The solution was washed at 0° C.first with a 5% aqueous solution (50 ml) of sodium carbonate and asaturated edible salt solution (50 ml) and the chloroform layer wasdried with anhydrous magnesium sulfate. The solvent was eliminated bydistillation and the residue was purified by way of columnchromatography (chloroform) through silica gel (7 g) followed byrecrystallization from n-hexane-chloroform whereby 79 mg (29.6%) of7-octanoyloxymethylcamptothecin were obtained as light yellow whiteneedle crystals. M.P. 188°-190° C.

IR_(max) ^(KBr) νcm⁻¹ : 3350, 2910, 1740, 1655, 1590, 1155, 1050, 765.

NMR (in CDCl₃) δ ppm: 1.04(3H, t, J=7.5 Hz), 1.24(13H, bs), 1.90(2H, q,J=7.5 Hz), 2.40(2H, t, J=7 Hz), 5.28 (2H, d, J=17 Hz), 5.45(2H, s),5.67(2H, s), 5.71(2H, d, J=17 Hz), 7.61(1H, s), 7.53-8.28(4H, m).

Ms: m/e 504 [M⁺ ] for C₂₉ H₃₂ N₂ O₆ =504.22.

EXAMPLE 15 (Preparation of 7-decanoyloxymethylcamptothecin)

7-Hydroxymethylcamptothecin (200 mg, 0.53 m-mol) was dissolved in warmedanhydrous pyridine (50 ml). n-Decanoyl chloride (300 mg, 3 eq) was addedto the solution and the mixture was stirred for 2 hours at 80° C.Thereafter, the solvent was removed by distillation under reducedpressure and the residue was dissolved in chloroform (100 ml). Thesolution was washed at 0° C. with a 5% aqueous solution (50 ml) ofsodium carbonate and saturated aqueous edible salt solution (50 ml) andthe chloroform layer was dried with anhydrous magnesium sulfate. Thesolvent was removed by distillation and the residue was purified by wayof column chromatography (chloroform) through silica gel (7 g) followedby recrystallization from n-hexane-chloroform whereby 83 mg (29.5%) of7-decanoyloxymethylcamptothecin were obtained as light yellow needlecrystals. M.P. 219°-221° C.

IR_(max) ^(KBr) νcm⁻¹ : 3220, 2910, 1730, 1655, 1590, 1160, 760.

NMR (in CDCl₂) δ ppm: 1.05(3H, t, 7.5 Hz), 1.22(17H, s), 1.91(2H, q,J=7.5 Hz), 2.42(2H, t, J=7 Hz), 5.28(2H, d, J=17 Hz), 5.44(2H, s),5.68(2H, s), 5.73(2H, d, J=17 Hz), 7.63(1H, s), 7.56-8.25(4H, m).

MS: m/e 532 [M⁺ ] for C₃₁ H₃₆ N₂ O₆ =532.26.

EXAMPLE 16 (Preparation of 7-isovaleroxymethylcamptothecin)

7-Hydroxymethylcamptothecin (200 mg, 0.53 m-mol) was dissolved in warmedanhydrous pyridine (50 ml). Isovaleric chloride (190 mg, 3 eq) was addedto the solution and the mixture was stirred for 2 hours at 80° C.Thereafter, the solvent was distilled off under reduced pressure and theresidue was dissolved in chloroform (100 ml). The solution was washed at0° C. with a 5% aqueous solution (50 ml) of sodium carbonate and asaturated aqueous solution (50 ml) of edible salt and the chloroformlayer was dried with anhydrous magnesium sulfate. The solvent wasremoved by distillation and the residue was purified by way of columnchromatography (chloroform) through silica gel (7 g) followed byrecrystallization from n-hexane-chloroform whereby 110 mg (44.9%) of7-isovaleroxymethylcamptothecin were obtained as light yellow whitecrystals. M.P. 240°-242° C.

IR_(max) ^(KBr) νcm⁻¹ : 3450, 2950, 1740, 1650, 1595, 1160, 760.

NMR (in CDCl₃) δ ppm: 0.94(6H, d, J=7 Hz), 1.07(3H, t, J=7 Hz), 1.91(2H,q, J=7 Hz), 2.31(2H, d, J=7 Hz), 3.71(1H, m), 5.30(2H, d, J=16 Hz),5.45(2H, s), 5.69(2H, d, J=16 Hz), 5.72(2H, s), 7.65(1H, s),7.57-8.27(4H, m).

MS: m/e 462 [M⁺ ] for C₂₆ H₂₆ N₂ O₆ =462.18.

EXAMPLE 17 (Preparation of 7-phenylacetoxymethylcamptothecin)

7-Hydroxymethylcamptothecin (500 mg, 1.32 m-mol) was dissolved in warmedanhydrous dimethylformamide (100 ml). Anhydrous pyridine (1 ml) andphenylacetyl choride (610 mg, 3 eq) were added to the solution and themixture was stirred for 2 hours at 80° C. Thereafter, the solvent wasremoved by distillation under reduced pressure and the residue wasdissolved in chloroform (200 ml). The solution was washed at 0° C. witha 5% aqueous solution (100 ml) of sodium carbonate and a saturatedaqueous solution (100 ml) of edible salt. The chloroform layer was driedwith anhydrous magnesium sulfate and the solvent was removed bydistillation. The residue was purified by way of column chromatography(chloroform) through silica gel (10 g) followed by recrystallizationfrom n-hexane-chloroform whereby (160 mg (24.4%) of7-phenylacetoxymethylcamptothecin were obtained as light yellow whitecrystals. M.P. 252°-253° C.

IR_(max) ^(KBr) νcm⁻¹ : 3400, 2970, 1760, 1655, 1600, 1160, 1130, 765.

NMR (in DMSO-d₆) δ ppm: 0.89(3H, t, J=7 Hz), 1.89(2H, q, J=7 Hz),3.77(2H, s) 5.42(2H, s), 5.45(2H, s), 5.79(2H, s), 7.25(5H, s), 7.35(1H,s), 7.60-8.30(4H, m).

MS: m/e 496 [M⁺ ] C₂₉ H₂₄ N₂ O₆ =496.16.

EXAMPLE 18 (Preparation of camptothecin-7-carboxylic acid)

7-Hydroxymethylcamptothecin (200 mg, 0.529 m-mol) was dissolved indioxane (300 ml). To this solution was added Jones reagent (2.5 ml,about 5.35 m-ml) and the mixture was stirred for 2 days at roomtemperature. The precipitate formed was filtered off and the filtratewas evaporated until dryness under reduced pressure. Water (15 ml) wasadded to the residue and an insoluble matter was collected on a filterand washed thoroughly with water (50 ml). This precipitate was purifiedby recrystallization from dioxane whereupon camptothecin-7-carboxylicacid was obtained as light yellow crystals having a melting point above300° C. The yield was 95 mg (45.8%).

IRν_(max) ^(KBr) cm⁻¹ : 3450-2670, 1760, 1650, 1595, 1240, 1170, 785.

NMR (CDCl₃) δ ppm: 0.91(3H, t, J=7 Hz), 1.90(2H, q, J=7 Hz), 5.31 (2H,s) 5.41(2H, s), 6.50(1H, br s, D₂ O ex.), 7.30(1H, s), 7.65-8.18(3H, m),8.76-8.84(1H, m).

MS m/e: 392.0995 [M⁺ ] (C₂₁ H₁₆ N₂ O₆ =392.1002).

UVν_(max) ^(EtOH) nm: 220, 249(sh), 290, 310, 336(sh), 360, 374.

EXAMPLE 19 (Preparation of ethyl camptothecin-7-carboxylate)

Camptothecin-7-carboxylic acid (40 mg, 0.102 m-mol) was suspended inethanol (15 ml). Concentrated sulfuric acid (0.5 ml) was added to thesuspension and the mixture was refluxed for 36 hours. The ethanol wasdistilled off under reduced pressure and ice water (100 ml) was added tothe residue to form a precipitate which was then collected byfiltration. On the other hand, a 5% aqueous solution of potassiumcarbonate was added in small portions to the filtrate to make the liquidneutral. The liquid was extracted with chloroform (100 ml×3) and theextract was dried with anhydrous magnesium sulfate, filtered andevaporated until dryness under reduced pressure. This residue wascombined with the precipitate already collected and purified byrecrystallization from ethanol-dioxane whereby ethylcamptothecin-7-carboxylate was obtained as yellow prismatic crystalshaving a melting point above 300° C. The yield was 26.5 mg(62%).

IRν_(max) ^(KBr) cm⁻¹ : 2930, 1775, 1750, 1660, 1590, 1230, 785.

NMR (DMSO-d₆) ppm: 0.91(3H, t, J=7.5 Hz), 1.35(3H, t, J=7.5 Hz),1.90(2H, q, J=7.5 Hz), 4.21(2H, q, J=7.5 Hz), 5.34(2H, s), 5.43(2H, brs), 7.37(1H, s), 7.70-8.70(4H, m).

MS m/e: 420 [M⁺ ] (C₂₃ H₂₀ N₂ O₆ =420.).

EXAMPLE 20 (Preparation of 5-methoxycamptothecin)

5-Hydroxycamptothecin (224 mg, 0.615 m-mol) was dissolved in methanol(40 ml). Boron trifluoride etherate (515 mg, 3.63 m-mol) was added tothe solution and the mixture was refluxed for 18 hours. The methanol wasremoved by distillation under reduced pressure and the residue wasshaken with water (100 ml) and further with chloroform (100 ml). Theaqueous phase in this case was then extracted with chloroform (100 ml×2)and the chloroform layer was combined with that already obtained in thepreceding treatment, dried with anhydrous magnesium sulfate, filteredand evaporated until dryness under reduced pressure. The residue wassubjected to column chromatography (2% methanolchloroform) throughsilica gel (50 g) to effect separation and purification of the productwhereby 159 mg (91.9%) of 5-methoxycamptothecin were obtained as ayellow white solid. Separately, 54 mg of 5-hydroxycamptothecin wererecovered.

IRν_(max) ^(KBr) cm⁻¹ : 3400, 2980, 1750, 1660, 1600, 1460, 1235, 1165.

NMR (CDCl₃) δ ppm: 1.02(3H, t, J=7.5 Hz), 1.92(2H, q, J=7.5 Hz),3.52(1.5H, s), 3.68(1.5H, s), 5.32.5.72(two 1Hs, dxd, J=19 Hz),6.75(0.5H, s), 6.89(0.5H, s) 7.60(1H, br s), 7.50-8.40(4H, m), 8.41(1H,br s).

MS m/e 378.1214 [M⁺ ] (C₂₁ H₁₈ N₂ O₅ =378.1209).

UVλ_(max) ^(EtOH) nm: 249(sh), 258, 297, 337(sh), 356, 372(sh).

EXAMPLE 21 (Preparation of 5-n-butoxycamptothecin)

5-Hydroxycamptothecin (160 mg, 0.439 m-mol) was dissolved in n-butanol(20 ml). Boron trifluoride etherate (1 ml) was added to the solution andthe mixture was refluxed for 1.5 hours. The reaction mixture wasevaporated until dryness under reduced pressure and the residue wasshaken with water (100 ml) and further with chloroform (150 ml). Thechloroform layer was dried with anhydrous magnesium sulfate, filteredand evaporated until dryness under reduced pressure. The residue wassubjected to column chromatography (chloroform) through silica gel (30g) to effect separation and purification of the product whereby5-n-butoxycamptothecin was obtained as a light yellow solid. The yieldwas 121 mg (65.5%).

IRν_(max) ^(KBr) cm⁻¹ : 3400, 2960, 1765, 1670, 1620, 1465, 1420, 1165.

NMR (CDCl₃ -DMSO-d₆) δ ppm: 1.02(3H, t, J=7.5 Hz), 0.86-1.80(7H, br m),1.94(2H, q, J=7.5 Hz), 4.00(2H, m), 5.22, 5.69(two 1Hs, dxd, J=17.5 Hz),6.77(0.5H, s), 6.87(0.5H, s), 7.33-8.40(4H, m), 7.61(1H, s), 8.41(1H,s).

EXAMPLE 22 (Preparation of 20-O-acetyl-5-acetoxycamptothecin)

5-Hydroxycamptothecin (30 mg, 0.082 m-mol) was dissolved in pyridine (10ml). Acetic anhydride (16 mg, 0.153 m-mol) was added to the solution andthe mixture was stirred for 3 hours at room temperature. The reactionmixture was evaporated until dryness under reduced pressure and theresidue was shaken with water (50 ml) and further with chloroform (50ml). The chloroform layer was dried with anhydrous magnesium sulfate,filtered and evaporated until dryness under reduced pressure. Theresidue was subjected to thin layer chromatography (2%methanol-chloroform) to effect separation of the products whereby 14 mg(41.8%) of an isomer of 5-acetoxycamptothecin having an Rf value of0.15, 12 mg (35.9%) of another isomer having an Rf value of 0.20 and 8mg (21.7%) of 20-O-acetyl-5-acetoxycamptothecin were obtained.

(1) 5-acetoxycamptothecin having an Rf value of 0.15:

M.P. 202°-205° C. (n-hexane-chloroform).

IRν_(max) ^(KBr) cm⁻¹ : 3400, 1760, 1670, 1620, 1160.

NMR (CDCl₃) δ ppm: 1.06(3H, t, J=7 Hz), 1.91(2H, q, J=7 Hz), 2.195(3H,s), 5.22, 5.65(two 1Hs, dxd, J=16.3 Hz), 7.62(1H, s), 7.91(1H, s),7.68-8.26(4H, m), 8.45(1H, s).

UVλ_(max) ^(EtOH) nm: 215, 223(sh), 252, 257, 297, 340(sh), 357, 372.

MS m/e: 406.1176 [M⁺ ] (C₂₂ H₁₈ N₂ O₆ =406.1158.).

[α]_(D) ²⁵ =+117.3° (C=5.2×10⁻³, EtOH).

(2) 5-acetoxycamptothecin having an Rf value of 0.20:

M.P. 258°-261° C. (n-hexane-chloroform).

IRν_(max) ^(KBr) cm⁻¹ : 3400, 1760, 1670, 1625, 1165.

NMR (in CDCl₃) δ ppm: 1.04(3H, t, J=7 Hz), 1.90(2H, q, J=7 Hz),2.192(3H, s), 5.22, 5.67(two 1Hs, dxd, J=16.6 Hz), 7.62(1H, s), 7.96(1H,s), 7.70-8.30(4H, m), 8.46(1H, s).

UVλ_(max) ^(EtOH) nm: 215, 223(sh), 252, 257, 296, 340(sh), 357,372(sh).

MS m/e: 406.1134 [M⁺ ].

[α]_(D) ²⁵ =-123° (C=3.33×10⁻³, EtOH).

(3) 20-O-acetyl-5-acetoxycamptothecin:

Rf=0.30

IRν_(max) ^(KBr) cm⁻¹ : 2930, 1765(vs), 1670, 1625, 1240.

NMR (in CDCl₃) δ ppm: 1.00(3H, m), 2.00(2H, m), 2.10(3H, s), 2.19(1.5H,s), 2.25(1.5H, s), 5.30 (0.5H, d, J=19 Hz), 5.31(0.5H, d, J=19 Hz),5.61(1H, d, J=19 Hz), 7.10(0.5H, s), 7.12(0.5H, s), 7.50-8.30(5H, m),8.50(1H, br s).

EXAMPLE 23 (Preparation of 5-benzoyloxycamptothecin)

5-Hydroxycamptothecin (200 mg, 0.549 m-mol) was dissolved in pyridine(10 ml). To this solution was added under agitation benzoyl chloride(180 mg, 1.28 m-mol) in small portions over 5 hours at room temperature.After addition of the benzoyl chloride, the agitation was continued forone hour. The reaction mixture was then evaporated until dryness underreduced pressure and the residue was taken up in water (50 ml).Insoluble matters were collected by filtration and dissolved inchloroform (100 ml) and the chloroform solution was dried with anhydrousmagnesium sulfate, decolored with active carbon, filtered and evaporateduntil dryness under reduced pressure. The residue was purified byrecrystallization from n-hexane-chloroform whereupon5-benzoyloxycamptothecin was obtained as light yellow prismaticcrystals. The yield was 154 mg (60%).

IRν_(max) ^(KBr) cm⁻¹ : 3400, 3080, 2980, 1760, 1740, 1670, 1610, 1460,1410, 1270.

NMR (CDCl₃ -DMSO-d₆) δ ppm: 1.02(3H, br t, J=7.5 Hz), 2.00(2H, br q,J=7.5 Hz), 5.23, 5.56(two 1Hs, dxd, J=17.5 Hz), 7.33-8.40(9H, m),7.53(1H, s), 7.21(0.5H, s), 7.32(0.5H, s), 8.72(1H, s).

MS m/e: 468.1296 [M⁺ ] (C₂₇ H₂₀ N₂ O₆ =468.1314.).

EXAMPLE 24 (Preparation of 7-methylcamptothecin)

Ferrous sulfate heptahydrate (4.17 g, 15 m-mol) and ethanol (3 ml, 60m-mol) were dissolved in water (30 ml). Camptothecin (700 mg, 2 m-mol)was suspended in the solution and dissolved therein by addingconcentrated sulfuric acid (15 ml) in small portions to the suspension.To the mixture was added dropwise under ice-cooling and agitation a 30%aqueous solution of hydrogen peroxide (1.63 ml, 16 m-mol). Afteraddition of the hydrogen peroxide, the mixture was stirred for 6 hoursat room temperature. To the reaction mixture was added ferrous sulfateheptahydrate (2.0 g, 7.2 m-mol) and then was added dropwise underice-cooling and agitation a 30% aqueous solution of hydrogen peroxide (1ml, 9.8 m-mol). The agitation was continued for 15 hours at roomtemperature. To complete the reaction, additional ferrous sulfateheptahydrate (4.2 g, 15 m-mol) and a 30% aqueous solution of hydrogenperoxide (1.5 ml, 14.7 m-mol) were added to the reaction mixture and thewhole was stirred for 8 hours at room temperature. The reaction mixturewas diluted with ice water (2.5 liters) and extracted with chloroform (3liters). The chloroform layer was dried with anhydrous magnesiumsulfate, filtered and evaporated until dryness under reduced pressure.The residue was purified by way of column chromatography (chloroform)through silica gel (10 g) followed by recrystallization fromn-hexane-chloroform whereby 127 mg (17.5%) of the objective compoundwere obtained as yellow needle crystals. M.P. 280°-281° C.

IRν_(max) ^(KBr) cm⁻¹ : 3350, 2920, 1755, 1650, 1600, 1470, 1155, 765.

NMR (in DMSO-d₆) δ ppm: 0.91(3H, t, J=7.5 Hz), 1.88(2H, q, J=7.5 Hz),2.79(3H, s), 5.26(2H,s), 5.41(2H, s), 6.43(1H, s, D₂ O,ex), 7.34(1H, s),7.57-8.32 (4H, m).

MS m/e: 362 [M⁺ ] (C₂₁ H₁₈ N₂ O₄ =362.37).

EXAMPLE 25 (Preparation of 7-methylcamptothecin)

Ferrous sulfate heptahydrate (2.0 g, 7 m-mol) was dissolved in water (15ml) and acetic acid (1.5 ml, 25 m-mol) was added thereto. Camptothecin(500 mg, 1.43 m-mol) was suspended in the resultant solution anddissolved therein by addition of concentrated sulfuric acid (8 ml) insmall portions. To this solution was added dropwise under ice-coolingand agitation tert-butyl hydroperoxide (900 mg, 10 m-mol) in smallportions. After addition of the tert-butyl hydroperoxide, the agitationwas continued for one hour at room temperature. The reaction mixture wasdiluted with ice water (500 ml) and extracted with chloroform (1.5 l).The chloroform layer was dried with anhydrous magnesium sulfate,filtered and evaporated until dryness under reduced pressure. Theresidue was washed thoroughly with acetone to obtain 440 mg (84.4%). Thecrude crystals were purified by recrystallization from pyridine-methanolwhereby 300 mg (57.6%) of the objective compound were obtained as lightyellow white needle crystals.

The analytical data of this product were identical with those of thecompound obtained in Example 24.

EXAMPLE 26 (Preparation of 7-ethylcamptothecin)

In an aqueous solution of sulfuric acid (15 ml of concentrated sulfuricacid in 30 ml of water) were dissolved camptothecin (1.00 g, 2.87m-mol), ferrous sulfate heptahydrate (5.60 g, 20.1 m-mol) and 1-propanol(6 ml, 86.1 m-mol). To this solution was added dropwise underice-cooling and agitation a 30% aqueous solution of hydrogen peroxide(2.1 ml, 20.1 m-mol) in small portions. After addition of the hydrogenperoxide, the agitation was continued for one hour at room temperature.The reaction was diluted with ice water (2 l) and extracted withchloroform (2.5 l). The chloroform layer was dried with anhydrousmagnesium sulfate, filtered and evaporated until dryness under reducedpressure. The residue was subjected to column chromatography(chloroform) through silica gel (15 g) to effect separation andpurification of the product which was then recrystallized fromn-hexane-chloroform whereby 265 mg (25.3%) of the objective compoundwere obtained as light yellow white needle crystals. M.P. 258°-261° C.

IRν_(max) ^(KBr) cm⁻¹ : 3370, 2920, 1750, 1650, 1600, 1460, 1150, 760.

NMR (in DMSO-d₆ -CDCl₃) δ: 0.97(3H, t, J=7 Hz), 1.39(3H, t, J=7 Hz),1.91(2H, q, J=7 Hz), 3.21(2H, q, J=7 Hz), 5.21(2H, s), 5.24(1H, d, J=16Hz), 5.57 (1H, d, J=16 Hz), 7.49(1H, s), 7.44-8.21 (4H, m).

MS m/e: 376.1399 [M⁺ ] (C₂₂ H₂₀ N₂ O₄ =376.1422).

EXAMPLE 27 (Preparation of 7-ethylcamptothecin)

Ferrous sulfate heptahydrate (350 mg, 1.25 m-mol) was dissolved in water(10 ml). Propionaldehyde (144 mg, 2.48 m-mol) was added to the solutionand dissolved therein by addition of acetic acid (10 ml). Camptothecin(175 mg, 0.5 m-mol) was suspended in the solution and then dissolvedtherein by addition of concentrated sulfuric acid (2 ml) in smallportions. To this solution was added dropwise under ice-cooling andagitation a 30% aqueous solution of hydrogen peroxide (144 mg, 1.27m-mol) in small portions. The agitation was continued for 15 minutesunder ice-cooling. The reaction mixture was diluted with ice water (500ml) and then extracted with chloroform (800 ml). The chloroform layerwas dried with magnesium sulfate, filtered and evaporated until drynessunder reduced pressure. The residue was subjected to columnchromatography (chloroform) through silica gel (10 g) to effectseparation and purification of the product whereby 105 mg (55.8%) of theobjective compound were obtained as light yellow white crystals. Theanalytical data of the resultant product were identical with those ofthe product obtained in Example 26.

EXAMPLE 28 (Preparation of 7-ethylcamptothecin)

Ferrous sulfate heptahydrate (1.0 g, 3.59 m-mol) was dissolved in water(10 ml). Diethyl ketone (1.29 g, 15 m-mol) was added to this solutionand acetic acid (6 ml) was then dissolved therein. Camptothecin (175 mg,0.5 m-mol) was suspended in this solution and then dissolved by additionof concentrated sulfuric acid (2 ml). To this solution was then addeddropwise under ice-cooling and agitation a 30% aqueous solution ofhydrogen peroxide (560 mg, 5 m-mol) in small portions. After addition ofthe hydrogen peroxide, the mixture was agitated for 48 hours at roomtemperature. The reaction mixture was diluted with ice water (500 ml)and extracted with chloroform (500 ml). The chloroform layer was driedwith magnesium sulfate, filtered and evaporated until dryness underreduced pressure. The residue was subjected to column chromatography(chloroform) through silica gel (10 g) to effect separation of theproduct whereby 17 mg (13.8%) of the objective compound and 61 mg ofunreacted camptothecin were obtained.

The analytical data of the resultant 7-ethylcamptothecin were identicalwith those of the compound obtained in Example 26.

EXAMPLE 29 (Preparation of 7-ethylcamptothecin)

Ferrous sulfate heptahydrate (1.20 g, 4.31 m-mol) and propionic acid wasdissolved in water (15 ml). Camptothecin (300 mg, 0.862 m-mol) wassuspended in the solution and dissolved therein by addition ofconcentrated sulfuric acid (6 ml) in small portions. To this solutionwas added dropwise under ice-cooling and agitation a 30% aqueoussolution of hydrogen peroxide (1 ml, 9.81 m-mol) in small portions overthe period of about 10 minutes. After addition of the hydrogen peroxide,the agitation was continued for 16 hours at room temperature. Thereaction mixture was diluted with ice water (300 ml) and extracted withchloroform (400 ml). The chloroform layer was dried with magnesiumsulfate, filtered and evaporated until dryness under reduced pressure.The residue was subjected to column chromatography (chloroform) throughsilica gel (10 g) to effect separation and purification of the productwhereby 73 mg (22.5 g) of the objective compound were obtained as ayellow white solid.

The analytical data of this compound were identical with those of thecompound obtained in Example 26.

EXAMPLE 30 (Preparation of 7-propylcamptothecin)

Ferrous sulfate heptahydrate (2.8 g, 10.1 m-mol) and 1-butanol (3 ml, 43m-mol) were dissolved in water (30 ml). Camptothecin (500 mg, 1.43m-mol) was suspended in the solution and dissolved therein by additionof concentrated sulfuric acid (15 ml). To this solution was addeddropwise under ice-cooling and agitation a 30% aqueous solution ofhydrogen peroxide (1.1 ml, 10.1 m-mol) in small portions. The agitationwas continued for 4 hours at room temperature. The reaction mixture wasdiluted with ice water (1.5 l) and extracted with chloroform (2 l). Thechloroform layer was dried with magnesium sulfate, filtered andevaporated until dryness under reduced pressure. The residue wassubjected to column chromatography (chloroform) through silica gel (15g) to effect separation and purification of the product which was thenrecrystallized from n-hexane-chloroform whereby 110 mg (21.0%) of theobjective compound were obtained as light yellow white needle crystals.M.P. 260°-261° C.

IRν_(max) ^(KBr) cm⁻¹ : 3400, 2930, 1745, 1650, 1600, 1455, 1155, 760.

NMR (CDCl₃) δ: 1.03(3H, t, J=7 Hz), 1.08(3H, t, J=8 Hz), 1.25-2.05(4H,m), 3.26(2H, t, J=8 Hz), 5.24(2H, s), 5.52(2H, dxd, J=17 Hz), 7.67(1H,s), 7.55-8.29(4H, m).

MS m/e: 390 [M⁺ ] (C₂₅ H₂₂ N₂ O₄ =390.43).

EXAMPLE 31 (Preparation of 7-propylcamptothecin)

Ferrous sulfate heptahydrate (800 mg, 2.88 m-mol) was dissolved in water(10 ml). Butyraldehyde (260 mg, 3.61 m-mol) was added to the solutionand dissolved therein by the addition of acetic acid (17 ml).Camptothecin (500 mg, 1.44 m-mol) was suspended in the solution anddissolved therein by the addition of concentrated sulfuric acid (2 ml)in small portions. To this solution was added dropwise under ice-coolingand agitation a 30% aqueous solution of hydrogen peroxide (333 mg, 2.93m-mol) in small portions. The agitation was continued for 20 minutesunder ice-cooling. The reaction mixture was diluted with ice water (1 l)and extracted with chloroform (1 l). The chloroform layer was dried withmagnesium sulfate, filtered and evaporated until dryness under reducedpressure. The residue was subjected to column chromatography(chloroform) through silica gel (15 g) to effect separation andpurification of the product whereby 333 mg (59.3%) of the objectivecompound was obtained as yellow white crystals. Purification of thecrude product by recrystallization from ethanol gave light yellowprismatic crystals. The analytical data of this product were identicalwith those of the product obtained in Example 30.

EXAMPLE 32 (Preparation of 7-butylcamptothecin)

Ferrous sulfate heptahydrate (3.0 g, 10.7 m-mol) was dissolved in water(30 ml). Camptothecin (500 mg, 1.43 m-mol) was suspended in the solutionand dissolved therein by addition of concentrated sulfuric acid (20 ml).To this solution was added 1-amyl alcohol (4.6 ml, 43 m-mol) and thenwas added dropwise under ice-cooling and agitation a 30% aqueoussolution of hydrogen peroxide (1.1 ml, 10.7 m-mol) in small portions.After addition of the hydrogen peroxide, the agitation was continued for4 hours at room temperature. The reaction mixture was diluted with icewater (1.5 l) and extracted with chloroform (2 l). The chloroform layerwas dried with magnesium sulfate, filtered and evaporated until drynessunder reduced pressure. The residue was subjected to columnchromatography (chloroform) through silica gel (10 g) to effectseparation and purification of the product which was further purified byrecrystallization from n-hexane-chloroform whereby 54 mg (9.3%) of theobjective compound was obtained as light yellow white needle crystals.M.P. 206°-207° C.

IRν_(max) ^(KBr) cm⁻¹ : 3350, 2930, 1745, 1680, 1600, 1450, 1150, 755.

NMR (CDCl₃) δ: 0.80-2.04(12H, m) 3.14(2H, t, J=7 Hz), 5.20(2H, s),5.26(1H, d, J=17 Hz), 5.73(1H, d, J=17 Hz), 7.62(1H, s), 7.28-7.88(2H,m), 7.96-8.26(2H, m).

MS m/e: 404 [M⁺ ] (C₂₄ H₂₄ N₂ O₄ =404).

EXAMPLE 33 (Preparation of 7-butylcamptothecin)

Ferrous sulfate heptahydrate (300 mg, 1.07 m-mol) was dissolved in water(10 ml). This solution was overlaid with n-amyl alcohol (310 μl, 2.86m-mol) and dimethylformamide (6 ml) was added to dissolve the n-amylalcohol in the solution. Camptothecin (50 mg, 0.143 m-mol) was suspendedin the solution and dissolved by the addition of concentrated sulfuricacid (1.5 ml). To this solution was added dropwise under ice-cooling andagitation a 30% aqueous solution of hydrogen peroxide (100 μl, 1.07m-mol) in small portions. After addition of the hydrogen peroxide, themixture was stirred for 4 hours at room temperature. The reactionmixture was diluted with ice water (100 ml) and extracted withchloroform (300 ml). The chloroform layer was dried with magnesiumsulfate, filtered and evaporated until dryness under reduced pressure.The residue was subjected to column chromatography through silica gel (4g) to effect separation and purification of the product which wasfurther purified by recrystallization from n-hexane-chloroform whereby18 mg (32%) of the objective compound were obtained as light yellowwhite needle crystals. The analytical data of this compound wereidentical with those of the compound obtained in Example 32.

EXAMPLE 34 (Preparation of 7-heptylcamptothecin)

Ferrous sulfate heptahydrate (800 mg, 2.88 m-mol) was dissolved in water(10 ml). Octanal (459 mg, 3.56 m-mol) was added to the solution anddissolved therein by the addition of acetic acid (20 ml). Camptothecin(500 mg, 1.44 m-mol) was suspended in this solution and dissolvedtherein by adding portionwise concentrated sulfuric acid (4 ml) to thesuspension. To this solution was added dropwise under ice-cooling andagitation a 30% aqueous solution of hydrogen peroxide (333 mg, 2.94m-mol) in small portions. After addition of the hydrogen peroxide, theagitation was continued for 15 minutes under ice-cooling. The reactionmixture was diluted with ice water (1 l) and extracted with chloroform(600 ml). The chloroform layer was dried with magnesium sulfate,filtered and evaporated until dryness under reduced pressure. Theresidue was subjected to column chromatography (chloroform) throughsilica gel (15 g) to effect separation and purification of the productwhereby 334 mg (53.5%) of the objective compound were obtained as ayellow white solid. Recrystallization of the solid from ethanol gaveyellow white needle crystals. M.P. 245°-246° C.

IRν_(max) ^(KBr) cm⁻¹ : 3380, 2920, 1750, 1655, 1600, 1460, 1160, 763.

NMR (in CDCl₃) δ: 0.80-2.05(18H, m), 3.16(2H, br, t, J=8 Hz), 5.23(2H,s) 5.30(1H, d, J=17 Hz), 5.72(1H, d, J=17 Hz), 7.65(1H, s),7.30-7.85(2H, m), 8.02-8.30(2H, m).

MS m/e: 446 [M⁺ ] (C₂₇ H₃₀ N₂ O₄ =446.22).

EXAMPLE 35 (Preparation of 7-nonylcamptothecin)

Ferrous sulfate heptahydrate (800 mg, 2.88 m-mol) was dissolved in water(10 ml). n-Decylaldehyde (560 mg, 2.94 m-mol) was added to the solutionand dissolved therein by the addition of acetic acid (32 ml).Camptothecin (500 mg, 1.44 m-mol) was suspended in the solution anddissolved therein by adding concentrated sulfuric acid (4 ml)portionwise to the suspension. To this solution was added dropwise underice-cooling and agitation a 30% aqueous solution of hydrogen peroxide(333 mg, 2.94 m-mol) in small portions. After addition of the hydrogenperoxide, the agitation was continued for 30 minutes under ice-cooling.The reaction mixture was diluted with ice water (1 l) and extractedwhich chloroform (1 l). The chloroform layer was dried with magnesiumsulfate, filtered and evaporated until dryness under reduced pressure.The residue was subjected to column chromatography (chloroform) throughsilica gel to effect separation and purification of the product whereby261 mg (38.3%) of the objective compound were obtained as a yellow whitesolid. Purification of this compound by recrystallization from methanolgave yellow white needle crystals. M.P. 205°-207° C.

IRν_(max) ^(KBr) cm⁻¹ : 3420, 2930, 1750, 1655, 1595, 1460, 1160, 762.

NMR (in CDCl₃) δ: 0.78-2.02(22H, m), 3.16(2H, br, t, J=7 Hz), 5.24(2H,s) 5.30(1H, d, J=17 Hz), 5.72(1H, d, J=17 Hz), 7.68(1H, s),7.50-7.90(2H, m), 8.02-8.30(2H, m).

MS m/e: 474 [M⁺ ] (C₂₉ H₃₄ N₂ O₄ =474.25).

EXAMPLE 36 (Preparation of 7-isobutylcamptothecin)

Ferrous sulfate heptahydrate (2.80 g, 10.1 m-mol) was dissolved in water(30 ml). The solution was overlaid with isoamyl alcohol (3.5 ml, 39m-mol) and dimethylformamide (10 ml) was added to dissolve the isoamylalcohol in the solution. Camptothecin (500 mg, 1.43 m-mol) was suspendedin this solution and dissolved therein by adding concentrated sulfuricacid (15 ml) to the suspension. To this solution was added dropwiseunder ice-cooling and agitation a 30% aqueous solution of hydrogenperoxide (1.1 ml, 10.1 m-mol) in small portions. After addition of thehydrogen peroxide, the agitation was continued for 40 minutes at roomtemperature. The reaction was diluted with ice water (1.5 l) andextracted with chloroform (1.5 l). The chloroform layer was dried withmagnesium sulfate, filtered and evaporated until dryness under reducedpressure. The residue was subjected to column chromatography(chloroform) through silica gel (15 g) to effect separation andpurification of the product which was further purified byrecrystallization from n-hexane-chloroform whereby 95 mg (16.7%) of theobjective compound were obtained as light yellow white needle crystals.M.P. 198°-200° C.

IRν_(max) ^(KBr) cm⁻¹ : 3400, 2930, 1740, 1650, 1595, 1450, 1155, 760.

NMR (CDCl₃) δ: 1.07(3H, t, J=7 Hz), 1.07(6H, d, J=7 Hz), 1.93(2H, q, J=7Hz), 2.12-2.40(1H, m), 3.09(2H, d, J=7 Hz), 5.28(2H, s), 5.54(2H, dxd,J=17 Hz), 7.68(1H, s), 7.55-8.29(4H, m).

MS: m/e 404 [M⁺ ] (C₂₄ H₂₄ N₂ O₄ =404).

EXAMPLE 37 (Preparation of 7-benzylcamptothecin)

Ferrous sulfate heptahydrate (3.40 g, 12.2 m-mol) was dissolved in water(30 ml). The solution was overlaid with β-phenethyl alcohol (3.20 g,28.6 m-mol) and acetic acid (27 ml) was added to dissolve theβ-phenethyl alcohol in the solution. Camptothecin (500 mg, 1.43 m-mol)was suspended in the solution and dissolved therein by the addition ofconcentrated sulfuric acid (30 ml) to the suspension. To this mixturewas added dropwise under ice-cooling and agitation a 30% aqueoussolution of hydrogen peroxide (1.5 ml, 14.7 m-mol) in small portions.After addition of the hydrogen peroxide, the agitation was continued for16 hours at room temperature. The reaction mixture was diluted with icewater (1.5 l) and extracted with chloroform (1.5 l). The chloroformlayer was dried with magnesium sulfate, filtered and evaporated untildryness under reduced pressure. The residue was washed thoroughly withn-hexane and subjected to column chromatography (chloroform) throughsilica gel (10 g) to effect separation and purification of the productwhich was further purified by recrystallization from n-hexane-chloroformwhereby 202 mg (50.6%) of the objective compound were obtained as lightyellow white needle crystals. M.P. 263°-265° C.

IRν_(max) ^(KBr) cm⁻¹ : 3360, 2800, 1735, 1650, 1590, 1440, 1145, 755,695.

NMR (in CDCl₃) δ: 1.03(3H, t, J=7.5 Hz), 1.89(2H, q, J=7.5 Hz), 4.58(2H,s), 5.14(2H, s), 5.26(1H, d, J=16.2 Hz), 5.73(1H, d, J=16.2 Hz),7.00-7.34(5H, m), 7.68(1H, s), 7.55-8.32(4H, m).

MS: m/e 438 [M⁺ ] (C₂₇ H₂₂ N₂ O₄ =438.47).

EXAMPLE 38 (Preparation of 7-β-phenethylcamptothecin)

In an aqueous solution of sulfuric acid (10 ml of concentrated sulfuricacid in 25 ml of water) were dissolved camptothecin (350 mg, 1 m-mol)and ferrous sulfate heptahydrate (2.0 g, 7.2 m-mol). The solution wasoverlaid with 3-phenylpropanol (1.5 g, 11.0 m-mol) and dimethylformamide(10 ml) was added to dissolve the 3-phenylpropanol in the solution. Tothis mixture was added dropwise under ice-cooling and agitation a 30%aqueous solution of hydrogen peroxide (740 μl, 7.2 m-mol) in smallportions. After addition of the hydrogen peroxide, the mixture wasstirred for 20 hours at room temperature. To this reaction mixture wereadded ferrous sulfate heptahydrate (2.0 g, 7.2 m-mol), 3-phenylpropanol(1.5 g, 11.0 m-mol) and dimethylformamide (35 ml). To the mixture wasadded under ice-cooling and agitation a 30% aqueous solution of hydrogenperoxide (740 μl, 7.2 m-mol). The mixture was further stirred for 20hours at room temperature. The reaction mixture was diluted with icewater (1.5 l) and extracted with chloroform (2.0 l). The chloroformphase was dried with magnesium sulfate, filtered and evaporated untildryness under reduced pressure. The residue was subjected to columnchromatography (chloroform) through silica gel (10 g) to effectseparation and purification of the product which was further purified byrecrystallization from n-hexane-chloroform whereby 66 mg (14.2%) of theobjective compound were obtained as light yellow white needle crystals.M.P. 260°-262° C.

IRν_(max) ^(KBr) cm⁻¹ : 3370, 2920, 1745, 1655, 1600, 1450, 1155, 755,700.

NMR (in CDCl₃) δ: 1.02(3H, t, J=7.5 Hz), 1.89(2H, q, J=7.5 Hz), 3.47(2H,t, J=7 Hz), 3.80(2H, t, J=7 Hz), 4.78(2H, s), 5.24(1H, d, J=17 Hz),5.70(1H, d, J=17 Hz), 6.98-7.40(5H, m), 7.61(1H, s), 7.51-8.38(4H, m).

MS: m/e 452 [M⁺ ] (C₂₈ H₂₄ N₂ O₄ =452.49).

EXAMPLE 39 (Preparation of 7-isopropylcamptothecin)

Ferrous sulfate heptahydrate (2.0 g, 7.5 m-mol) was dissolved in water(20 ml). The solution was overlaid with isobutanol (2.75 ml, 30 m-mol)and acetic acid (6 ml) was added to dissolve the isopropanol in thesolution. Camptothecin (350 mg, 1 m-mol) was suspended in the solutionand dissolved therein by addition of concentrated sulfuric acid (17 ml)to the suspension. To this solution was added dropwise under ice-coolingand agitation a 30% aqueous solution of hydrogen peroxide (760 μl, 7.5m-mol) in small portions. After addition of the hydrogen peroxide, themixture was stirred for 30 minutes at room temperature. The reactionmixture was diluted with ice water (1 l) and extracted with chloroform(1.5 l). The chloroform layer was dried with magnesium sulfate, filteredand evaporated until dryness under reduced pressure. The residue wassubjected to column chromatography (chloroform) through silica gel (10g) to effect separation and purification of the product which wasfurther purified by recrystallization from n-hexane-chloroform whereby128 mg (32.8%) of the objective compound were obtained as light yellowwhite needle crystals. M.P. 258°-259° C.

IRν_(max) ^(KBr) cm⁻¹ : 3400, 2950, 1750, 1645, 1595, 1460, 1155, 760.

NMR (in CDCl₃) δ: 1.04(3H, t, J=7.5 Hz), 1.54(6H, d, J=7 Hz), 1.90(2H,q, J=32 7.5 Hz), 4.00(1H, heptet, J=7Hz), 5.29(1H, d, J=17 Hz), 5.37(2H,s), 5.75(1H, d, J=17 Hz), 7.63(1H, s), 7.45-8.36(4H, m).

MS: m/e 390 [M⁺ ] (C₂₃ H₂₂ N₂ O₄ =390).

EXAMPLE 40 (Preparation of 7-cyclohexyl camptothecin)

Ferrous sulfate heptahydrate (3.0 g, 10.73 m-mol) was dissolved in water(30 ml). The solution was overlaid with cyclohexylmethanol (1.63 ml,14.3 m-mol) and acetic acid (22 ml) was added with stirring to dissolvethe cyclohexylmethanol in the solution. Camptothecin (500 mg, 1.43m-mol) was suspended in this solution and dissolved therein by addingconcentrated sulfuric acid (8 ml) to the suspension. To this mixture wasadded dropwise under ice-cooling and agitation a 30% aqueous solution ofhydrogen peroxide (1.1 ml, 10.73 m-mol) in small portions. Afteraddition of the hydrogen peroxide, the agitation was continued for 30minutes at room temperature. The reaction mixture was diluted with icewater (1 l) and extracted with chloroform (1.5 l). The chloroform layerwas dried with magnesium sulfate, filtered, and evaporated until drynessunder reduced pressure. The residue was subjected to columnchromatography (chloroform) through silica gel (10 g) to effectseparation and purification of the product which was further purified byrecrystallization from n-hexane-chlorform whereby 181 mg (29.4%) of theobjective compound were obtained as light yellow white needle crystals.M.P. 260°-261° C.

IRν_(max) ^(KBr) cm⁻¹ : 3380, 2920, 1745, 1655, 1595, 1440, 1155, 765.

NMR (in CDCl₃) δ: 1.04(3H, t, J=8 Hz), 1.20-2.18(12H, m), 3.70(1H, m),5.30(1H, d, J=17 Hz), 5.39(2H, s), 5.72(1H, d, J=17 Hz), 7.67(1H, s),7.50-7.85(2H, m), 8.16-8.27(2H, m).

MS: m/e 430 [M⁺ ] (C₂₆ H₂₆ N₂ O₄ =430.19).

It is understood that the preceding representative examples may bevaried within the scope of the present specification, both as toreactants and reaction conditions, by one skilled in the art to achieveessentially the same results.

As many widely different embodiments of this invention may be madewithout departing from the spirit and scope thereof, it is to beunderstood that this invention is not limited to the specificembodiments thereof except as defined in the appended claims.

What is claimed is:
 1. A camptothecin derivative of the formula:##STR9## wherein X is H, CH₂ OH, COOH, a straight or branched chainalkyl group with 1-18 carbon atoms, a cycloalkyl group with 5-7 carbonatoms, a phenylalkyl group having 1 to 3 carbon atoms in the alkyleneportion thereof, or the group CH₂ OCOR¹ or COOR² where R¹ is a straightor branched chain alkyl group with 1-17 carbon atoms, trifluoromethyl,phenyl, a phenylalkyl group having 1 to 2 carbon atoms in the alkyleneportion thereof or the group HOOC--(CH₂)_(n) where n is an integer of 2to 4 and where R² is a lower alkyl group; Y is H, OH or OR³, where R³ isa lower alkyl group, an alkanoyl group with 1-5 carbon atoms, benzoyl ora phenylalkanoyl group with 1-2 carbon atoms in the alkylene portionthereof: and Z is H, acetyl or trifluoroacetyl; with the proviso (1)that when X is CH₂ OH, the straight or branched alkyl group, thecycloalkyl group or the phenylalkyl group, both Y and Z are H, (2) thatwhen X is CH₂ OCOR¹ or COOR², Y is H, (3) that when Y is OH, both X andZ are H, (4) that when Y is OR³, X is H, .Iadd.and (5) X and Y are notboth simultaneously hydrogen; .Iaddend.or a water-soluble alkali metalsalt thereof.
 2. 7-Hydroxymethylcamptothecin.
 3. 5-Hydroxycamptothecin.4. A camptothecin derivative of the formula:

    7-(R'CO--OCH.sub.2)-camptothecin

wherein R' is a straight or branched chain alkyl group with 1-10 carbonatoms, phenyl or a phenylalkyl group with 1-2 carbon atoms in thealkylene portion thereof or the group HOOC--(CH₂)_(n) where n is aninteger of 2 to
 4. 5. A camptothecin derivative of the formula:

    7-(R.sup.5 CO--OCH.sub.2)-20-O-(R.sup.5 CO)-camptothecin

wherein the two R⁵ 's are identical with each other and each representsmethyl or trifluoromethyl.
 6. A camptothecin derivative of the formula:

    7-R.sup.6 -camptothecin

wherein R⁶ is a straight or branched chain alkyl group with 1-10 carbonatoms or a cycloalkyl group with 5-7 carbon atoms.
 7. A camptothecinderivative of the formula:

    7-R.sup.7 -camptothecin

wherein R⁷ is phenylalkyl group having 1 to 3 carbon atoms in thealkylene portion thereof.
 8. A camptothecin derivative of the formula:

    5-(R.sup.8 O)-camptothecin

wherein R⁸ is a straight or branched chain alkyl group with 1-5 carbonatoms.
 9. A camptothecin derivative of the formula:

    5-(R.sup.9 COO)-camptothecin

wherein R⁹ is a straight or branched chain alkyl group with 1-4 carbonatoms, phenyl or a phenylalkyl group having 1 to 3 carbon atoms in thealkylene portion thereof.
 10. 5-Acetyloxy-20-O-acetylcamptothecin. 11.Camptothecin-7-carboxylic acid.
 12. A camptothecin derivative of theformula:

    7-(R.sup.10 --OOC)-camptothecin

wherein R¹⁰ is a straight or branched chain alkyl group with 1 to 4carbon atoms.
 13. A process for the preparation of a camptothecinderivative of the formula: ##STR10## wherein X' is the group COOR⁴ orCH₂ OR where R⁴ is H, lower alkyl or benzyl and R is H or R'CO where R'is a straight or branched chain alkyl group with 1-17 carbon atoms,trifluormethyl, phenyl, a phenylalkyl group having 1 to 2 carbon atomsin the alkylene portion thereof or the group HOOC(CH₂)_(n) where n is aninteger of 2 to 4, and Z is H, acetyl or trifluoroacetyl, or awater-soluble alkali metal salt thereof, which comprises subjectingcamptothecin to a radical reaction with a hydroxymethyl compound of thegeneral formula:

    A--CH.sub.2 OH                                             (II)

wherein A is H, COOH or CH₂ OH, by the aid of sulfuric acid and aninorganic or organic peroxide in an aqueous medium, the inorganic andorganic peroxide being selected from the group consisting of hydrogenperoxide, persulfuric acid and ammonium and alkali metal salts thereof,Caro's acid and alkali metal salts thereof, alkali metal and alkalineearth metal peroxides, tert-butyl hydroperoxide, benzoyl peroxide anddialkanoyl peroxides.
 14. A process according to claim 13, furthercomprising the step of treating the resultant7-hydroxymethylcamptothecin with a compound of the formula:

    R'COOH

wherein R' has the same meaning as defined above, or an anhydride oracid halide thereof, to give the 7-R'COOCH₂ group wherein R' is asdefined above with or without simultaneous acetylation ortrifluoroacetylation.
 15. A process according to claim 13, furthercomprising the step of oxidizing the resultant7-hydroxymethylcamptothecin with an oxidizer selected from the groupconsisting of chromium trioxide, alkali metal dichromates andpermanganates to 7-carboxycamptothecin.
 16. A process according to claim14 or 15, further comprising the step of esterifying the 7-carboxylgroup with a compound of the formula:

    R.sup.4 --OH

wherein R⁴ has the same meaning as defined above, to form a7-X"-camptothecin wherein X" is the group R⁴ OCO-- where R⁴ is asdefined above.
 17. A process according to claim 13, wherein the radicalreaction is carried out in the presence of a metal ion supplied in thereaction medium from a transition metal salt selected from the groupconsisting of silver, iron (II), copper, cobalt, nickel, lead, mercury,cadmium, thallium and zinc salts in the form of halides, carbonates,nitrates, sulfates and acetates.
 18. A process according to claim 17,wherein the transition metal salt is silver nitrate.
 19. A processaccording to claim 17, wherein the transition metal salt is ferroussulfate.
 20. A process according to claim 13, wherein the peroxide isammonium persulfate.
 21. A process according to claim 13, wherein theperoxide is hydrogen peroxide.
 22. A process according to claim 17,wherein the transition metal salt is used within the range from analmost equimolar amount to an about 30 molar amount with respect to theamount of camptothecin used.
 23. A process according to claim 13,wherein the reaction is carried out at a temperature varying from roomtemperature to the boiling point of the reaction mixture.
 24. A processaccording to claim 13, wherein the hydroxymethyl compound is methanol.25. A process for the preparation of a camptothecin derivative of theformula: ##STR11## wherein X'" is a straight or branched chain alkylgroup with 1-18 carbon atoms, a cycloalkyl group with 5-7 carbon atoms,a phenylalkyl group having 1 to 3 carbon atoms in the alkylene portionthereof, or the group --COOR⁴ where R⁴ is H or a lower alkyl group, or awater-soluble alkali metal salt thereof, which comprises subjectingcamptothecin to a radical reaction with an organic compound of thegeneral formula:

    X"--Q                                                      (II')

wherein Q is --CH₂ OH, --COOH, --CHO, --COX" or ##STR12## and X" is astraight or branched chain alkyl group with 1-10 carbon atoms, acycloalkyl group with 5-7 carbon atoms, or a phenylalkyl group having 1to 3 carbon atoms in the alkylene portion thereof, by the aid ofsulfuric acid and an inorganic or organic peroxide in an aqueous mediumin the presence of a transition metal ion supplied in the medium from atransition metal salt selected from the group consisting of silver, iron(II), copper, cobalt, nickel, lead, mercury, cadmium, thallium and zincsalts in the form of halides, carbonates, nitrates, sulfates andacetates, the inorganic or organic peroxide being selected from thegroup consisting of hydrogen peroxide, persulfuric acid and ammonium andalkali metal salts thereof, Caro's acid and alkali metal salts thereof,alkali metal and alkaline earth metal peroxides, tert-butylhydroperoxide, benzoyl peroxide and dialkanoyl peroxides.
 26. A processaccording to claim 25, further comprising the step of oxidizing theresultant 7-X"-camptothecin derivative wherein X" is an alkyl group withan oxidizing agent selected from the group consisting of chromiumtrioxide, alkali metal dichromates and permanganates to a7-X'"-camptothecin derivative wherein X'" is a carboxy group.
 27. Aprocess according to claim 26, further comprising the step ofesterifying the 7-carboxy group with a lower alkanol to form a7-X'"-camptothecin wherein X'" is a lower alkoxycarbonyl group.
 28. Aprocess according to claim 25, wherein the transition metal salt issilver nitrate.
 29. A process according to claim 25, wherein thetransition metal salt is ferrous sulfate.
 30. A process according toclaim 25, wherein the peroxide is ammonium persulfate.
 31. A processaccording to claim 25, wherein the peroxide is hydrogen peroxide.
 32. Aprocess according to claim 25, wherein the organic compound is used in alarge excess in molar ratio with respect to the camptothecin.
 33. Aprocess according to claim 32, wherein the organic compound is used inan amount of about 20 molar proportion with respect to the camptothecin.34. A process according to claim 25, wherein the transition metal saltand the peroxide are used respectively in an amount of about 5 to 8molar excess with respect to the camptothecin.
 35. A process accordingto claim 25, wherein the peroxide is tert-butyl hydroperoxide.
 36. Aprocess for the preparation of a camptothecin derivative of the formula:##STR13## wherein Y' is OH or the group OR³ where R³ is a lower alkylgroup, a lower alkylcarbonyl group, benzoyl or a phenylalkyl grouphaving 1 to 3 carbon atoms in the alkylene portion thereof, and Z is Hor acetyl, or a water-soluble alkali metal salt thereof, which comprisestreating camptothecin with sulfuric acid and a persulfate selected fromthe group consisting of ammonium persulfate, alkali metal persulfates,Caro's acid and alkali metal salts thereof in an aqueous mediumcontaining a transition metal ion supplied in the medium from atransition metal salt selected from the group consisting of silver, iron(II), copper, cobalt, nickel, lead, mercury, cadmium, thallium and zincsalts in the form of halides, carbonates, nitrates, sulfates andacetates.
 37. A process according to claim 36, further comprising thestep of treating the resultant 5-hydroxycamptothecin with a compound ofthe general formula:

    R.sup.3 --OH

wherein R³ has the same meaning as defined above, to convert the5-hydroxy group into a 5-R³ 'O group where R³ ' is a lower alkyl group,or into a 5-R³ "O group where R³ " is a lower alkylcarbonyl group, abenzoyl group or a phenylalkylcarbonyl group having 1 to 3 carbon atomsin the alkylene portion thereof, with or without simultaneousacetylation of the 20-hydroxy group.
 38. A process according to claim 36wherein the transition metal salt is silver nitrate.
 39. A processaccording to claim 36, wherein the transition metal salt is ferroussulfate.
 40. A process according to claim 36, wherein the persulfate isused in an amount within the range of 5-30 molar proportion with respectto the camptothecin.